Molecular Diagnostics

QUICK VISUAL REFERENCE FOR COMMON
TECHNIQUES IN MOLECULAR BIOLOGY
40.00
107 copies
106 copies 35.00
100 105 copies
30.00
104 copies
Threshold cycle (C)

103 copies 25.00
102 copies
Rn
10 20.00
101 copies Y = –3.345(x) + 38.808
R2 = 0.9983
15.00
1 10.00
5.00
0.1 0.00
1 3 5 7 9 21 23 25 27 29 33 35 37 39 41 43 45 47 49 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07
A Cycle B Starting quantity (copies/rxn)
■ Quantitative polymerase chain reaction (PCR)
A
Probes hybridized C
Cell nucleus G
to chromosomes
T
Normal cell (diploid) Triploid Deletion

■ Fluorescence in situ hybridization (FISH) analysis for normal
diploid cell (left), triploidy (center), and deletion (right)
G A T T C T G A A T T A G C T G T A T C G
C5 : YGYGTTTATGYGAGGTYGGGTGGGYGGGTYGTTAGTTTYG
0% 0% 1% 0% 1% 1% 1%
1200
1000
800
600
400 N N T T S T G N M A T Y N K C T K N A T C G
200 ■ Examples of good sequence quality (top)
0
–200 and poor sequence quality (bottom)
E SGT C TGT CGT A T AGT CGA TGT CGT AGT C TGT CGT A TGT T C
5 10 15 20 25 30 35
A4 : YGYGTTTATGYGAGGTYGGGTGGGYGGGTYGTTAGTTTYG
37% 1% 33% 38% 42% 46% 46%
1500
1000
500
E SGT C TGT CGT A T AGT CGA TGT CGT AGT C TGT CGT A TGT T C
5 10 15 20 25 30 35
■ DNA methylation at cytosine residues detected by pyrosequencing

of bisulfite-treated DNA Continued on inside back cover
Molecular
Diagnostics
Fundamentals, Methods,
and Clinical Applications
THIRD EDITION
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Molecular
Diagnostics
Fundamentals, Methods,
and Clinical Applications
THIRD EDITION
Lela Buckingham, PhD, MB (ASCP), DLM (ASCP)

College of Health Sciences
Rush University Medical Center
Chicago, Illinois
Acknowledgments
Thanks and gratitude are extended to all who helped This book was originally envisioned by my co-author
in the completion of the third edition of this work. The for the first edition, Dr. Maribeth Flaws. Thanks to her
useful input provided by reviewers who gave their valu- for initiating this project. Thanks to Dr. Herb Miller and
able time to comment on and improve the writing is the Medical Laboratory Science faculty in the Rush Uni-
gratefully acknowledged. Molecular laboratory science versity College of Health Sciences for the opportunity
is an example of innovative technology applied to the to participate in medical laboratory science education. I
ultimate goal of improved patient care. greatly appreciate the guidance and support of the pub-
I owe thanks to my colleagues at Rush University lication staff at F. A. Davis—Christa Fratantoro, Julie
Medical Center, Dr. Wei-Tong Hsu, Dr. Nick Moore, Chase, Roxanne Klaas, and Katharine Margeson—for
Dr. Mary Hayden, Dr. Sivadasan Kanangat, and Dr. Eliz- the illustration and production of the text.
abeth Berry-Kravis, for help and support in their areas of I would like to acknowledge and thank fellow
expertise. I would also like to acknowledge colleagues members of the Association for Molecular Pathology, a
in the Rush University College of Health Sciences, res- vibrant and resourceful organization dedicated to educa-
idents, fellows, students, and laboratory professionals tion and policy in the practice of molecular diagnostics.
who provided suggestions for the third edition, partic- This organization has provided an outlet for contex-
ularly Alexandra Vardouniotis, Dr. Mezgebe Gebrekiris- tual information, training, and sanction to further this
tos, and Adrian Tira, with whom I work and from whom ever-advancing field of study.
I learn every day.
xi
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Library of Congress Cataloging-in-Publication Data
Names: Buckingham, Lela, author.

Title: Molecular diagnostics : fundamentals, methods, and clinical
applications / Lela Buckingham.
Description: Third edition. | Philadelphia : F.A. Davis Company, [2019] |
Includes bibliographical references and index.
Identifiers: LCCN 2018058583 (print) | LCCN 2018059084 (ebook) |
ISBN 9780803699540 | ISBN 9780803668294 (alk. paper)
Subjects: | MESH: Molecular Diagnostic Techniques—methods | Nucleic
Acids—analysis | Genetic Techniques
Classification: LCC RB43.7 (ebook) | LCC RB43.7 (print) | NLM QY 102 |
DDC 616.9/041—dc23
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To our students committed to
service through the practice of Medical Laboratory Science
Preface
Molecular technology continues to grow in importance purpose, principles, and interpretation of the molecular
in the clinical laboratory. Training of health-care pro- diagnostic tests that they will be ordering and assessing
fessionals routinely includes molecular biology, from for their patients.
laboratory techniques to therapeutic decisions. This text- Students who are first learning about molecular-based
book was written to provide fundamental knowledge of assays will find the text useful for explaining the funda-
molecular biology, current methods, and their clinical mental principles. Practitioners who are performing and
applications. interpreting these assays can use this text as a resource
The primary audience for this text is students enrolled for reference and troubleshooting and to drive the imple-
in Clinical/Medical Laboratory Science programs at all mentation of additional molecular-based assays in their
levels. It explains the principles of molecular technol- laboratories.
ogy that are used for diagnostic purposes. Examples of An Instructor ’s Resource package has been devel-
applications of molecular-based assays are included in oped for educators who adopt this text for a course.
the text, along with case studies that illustrate the use These resources, including PowerPoint presentations, a
and interpretation of these assays in patient care. test-item bank, and additional case studies, are available
This text is also appropriate for those in other on DavisPlus at http://davisplus.fadavis.com.
health-related disciplines who need to understand the
Lela Buckingham
vii
Reviewers
Catherine E. Bammert, MS, CT, MB (ASCP)CM Rachel Hulse, MS, MLS (ASCP)CM
Associate Professor Program Director
Program Director, Diagnostic Molecular Science Medical Laboratory Sciences
Clinical Laboratory Sciences Idaho State University
Northern Michigan University Pocatello, Idaho
Marquette, Michigan
Marisa K. James, MA, MLS (ASCP)CM
Katie Bennett, PhD, MB (ASCP), NRCC-CC Program Director
Assistant Professor and Laboratory Director School of Clinical Laboratory Science
Laboratory Sciences and Primary Care North Kansas City Hospital
Texas Tech University Health Sciences Center North Kansas City, Missouri
Lubbock, Texas
Jacqueline Peacock, PhD, MB (ASCP)CM
Tammy Carter, PhD, MT (ASCP), MB (ASCP) Assistant Professor and Program Coordinator
Assistant Professor, CLS Program Director Clinical Laboratory, Respiratory Care, and Health
Laboratory Science and Primary Care Administration Programs
Texas Tech University Health Sciences Center Ferris State University
Lubbock, Texas Grand Rapids, Michigan
Kristen Coffey, MS David Petillo, PhD, MT (ASCP)CM, MB

Visiting Instructor Clinical Coordinator/Assistant Professor
Medical Laboratory Sciences College of Health Professions
University of West Florida Molecular Diagnostics Program
Pensacola, Florida Ferris State University
Grand Rapids, Michigan
Daniel Harrigan, MS, MB (ASCP)CM
Professor
Laboratory Sciences
Blackhawk Technical College
Monroe, Wisconsin
ix
x Reviewers
Linda M. Ray, MS (ASCP)CM EDITORIAL REVIEWERS

Assistant Professor
Medical Laboratory Science We especially thank our editorial reviewers for assisting
University of North Dakota, School of Medicine & with page proof review.
Health Sciences
Grand Forks, North Dakota Mezgebe Gebrekiristos, PhD, MS, MT (ASCP)
Clinical Laboratory Scientist
Barbara Sawyer, PhD, MLS (ASCP), MB Molecular Oncology Laboratory
Professor Pathology Department
Lab Sciences and Primary Care Rush University Medical Center
Texas Tech University Health Sciences Center Chicago, Illinois
Lubbock, Texas
Lenny K. Hong, M.S., MLS (ASCP)CM, MB (ASCP)CM
CM
Ebot Sahidu Tabe, BMLS, MS, PhD, MB (ASCP) PhD Graduate Student - Department of Pathology
Instructor University of Illinois at Chicago - College of Medicine
Basic and Clinical Sciences Chicago, Illinois
Albany College of Pharmacy and Health Sciences
Albany, New York Adrian Tira, MSc., MT (ASCP)
Department of Pathology
Geoffrey Toner, MS, MB (ASCP)CM Rush University Medical Center
Instructor/Education Coordinator Chicago, Illinois
Medical Laboratory Sciences and Biotechnology
Jefferson College of Health Professions Alexandra Vardouniotis MS, MLS (ASCP)CM SBBCM
Thomas Jefferson University Medical Laboratory Scientist
Philadelphia, Pennsylvania Rush University Medical Center
Chicago, Illinois
Contents
Transfer RNA 33
Section I Other RNAs 35
Fundamentals of Molecular Biology: RNA POLYMERASES 35
OTHER RNA-METABOLIZING ENZYMES 36
An Overview 1
Ribonucleases 36
1 Nucleic Acids and Proteins 2 RNA Helicases 37
PROTEINS AND THE GENETIC CODE 37
DNA 3
Amino Acids 38
DNA STRUCTURE 4
Genes 43
Nucleotides 4
The Genetic Code 43
Nucleic Acid 8
TRANSLATION 46
DNA REPLICATION 9
Amino Acid Charging 46
Polymerases 11
Protein Synthesis 47
ENZYMES THAT METABOLIZE DNA 14
Restriction Enzymes 14 2 Gene Expression and Epigenetics 57
DNA Ligase 17 TRANSCRIPTION 58
Other DNA Metabolizing Enzymes 17 Transcription Initiation 58
RECOMBINATION IN SEXUALLY REPRODUCING ORGANISMS 19 Transcription Elongation 58
RECOMBINATION IN ASEXUAL REPRODUCTION 21 Transcription Termination 59
Conjugation 21 REGULATION OF TRANSCRIPTION 60
Transduction 23 Regulation of Messenger RNA Synthesis at Initiation 60
Transformation 23 Post-Transcriptional Regulation 64
PLASMIDS 25 Post-Translational Regulation 64
RNA 27 EPIGENETICS 65
Transcription 27 Histone Modification 65
Transcription Initiation 28 Nucleic Acid Methylation 66
Transcription Elongation 28 CLASSIFICATION OF EPIGENETIC FACTORS 69
Transcription Termination 29 NONCODING RNAs 69
TYPES/STRUCTURES OF RNA 29 MicroRNAs 70
Ribosomal RNA 29 Small Interfering RNAs 70
Messenger RNA 29 Other Small RNAs 70
Small Nuclear RNA 33 Long Noncoding RNAs 71
xiii
xiv Contents
Protein Probes 124

Section II Probe Labeling 126
Common Techniques in Molecular Nucleic Acid Probe Design 126
HYBRIDIZATION CONDITIONS, STRINGENCY 128
Biology 77
DETECTION SYSTEMS 129
3 Nucleic Acid Extraction Methods 78 INTERPRETATION OF RESULTS 132
ISOLATION OF DNA 79 ARRAY-BASED HYBRIDIZATION 133
Preparing the Sample 79 Dot/Slot Blots 133
DNA Isolation Chemistries 81 Genomic Array Technology 134
ISOLATION OF RNA 87 SOLUTION HYBRIDIZATION 138
Total RNA 87 6 Nucleic Acid Amplification 142
Specimen Collection 87 TARGET AMPLIFICATION 143
RNA Isolation Chemistries 87 Polymerase Chain Reaction 143
MEASUREMENT OF NUCLEIC ACID QUALITY AND QUANTITY 90 Transcription-Based Amplification Systems 164
Electrophoresis 90 Genomic Amplification Methods 165
Spectrophotometry 92 PROBE AMPLIFICATION 168
Fluorometry 93 Ligase Chain Reaction 168
Microfluidics 94 Strand Displacement Amplification 169
4 Resolution and Detection of Nucleic Qβ Replicase 170
Acids 97 SIGNAL AMPLIFICATION 172
ELECTROPHORESIS OF NUCLEIC ACIDS 98 Branched DNA Amplification 172
Hybrid Capture Assays 173
GEL SYSTEMS 99
Cleavage-Based Amplification 173
Agarose Gels 99
Cycling Probe 174
Polyacrylamide Gels 101
CAPILLARY ELECTROPHORESIS 102 7 Chromosomal Structure and Chromosomal
BUFFER SYSTEMS 104 Mutations 179
Buffer Additives 105 CHROMOSOMAL STRUCTURE AND ANALYSIS 181
ELECTROPHORESIS EQUIPMENT 106 Chromosomal Compaction and Histones 181
Gel Loading 108 Chromosome Morphology 183
DETECTION SYSTEMS 109 Visualizing Chromosomes 184
Fluorescent Dyes 109 DETECTION OF GENOME AND CHROMOSOMAL MUTATIONS 186
Silver Stain 110 Karyotyping 186
Fluorescence In Situ Hybridization 190
5 Analysis and Characterization of Nucleic COMPARATIVE GENOME HYBRIDIZATION (CGH) 195
Acids and Proteins 112
RESTRICTION ENZYME MAPPING OF DNA 113
8 Gene Mutations 199
TYPES OF GENE MUTATIONS 200
CRISPR ENZYME SYSTEMS 115
DETECTION OF GENE MUTATIONS 201
HYBRIDIZATION TECHNOLOGIES 116
Biochemical Methods 201
Southern Blots 117
Nucleic Acid Analyses 207
PROBE HYBRIDIZATION 121
GENE VARIANT NOMENCLATURE 218
Northern Blots 122
GENE NAMES 219
Western Blots 122
PROBES 123 9 DNA Sequencing 223
DNA Probes 123 DIRECT SEQUENCING 224
RNA Probes 124 Manual Sequencing 224
Other Nucleic Acid Probe Types 124 Automated Fluorescent Sequencing 229
Contents xv
PYROSEQUENCING 235 QUALITY ASSURANCE 305

BISULFITE DNA SEQUENCING 236 Controls 305
RNA SEQUENCING 237 Quality Control 305
NEXT-GENERATION SEQUENCING 238 Selection of Sequence Targets for Detection
Gene Panels 240 of Microorganisms 307
NGS Library Preparation 240 MOLECULAR DETECTION OF MICROORGANISMS 308
Targeted Libraries 241 Bacteria 309
Sequencing Platforms 243 Viruses 313
Sequence Quality 246 Mycology 323
Filtering and Annotation 247 Parasites 324
BIOINFORMATICS 248 ANTIMICROBIAL AGENTS 324
THE HUMAN GENOME PROJECT 250 Resistance to Antimicrobial Agents 325
Variant Associations With Phenotype 253 Molecular Detection of Resistance 327
MOLECULAR EPIDEMIOLOGY 329
Molecular Strain Typing Methods for Epidemiological
Section III
Studies 330
Techniques in the Clinical Comparison of Typing Methods 336
Laboratory 259
12 Molecular Detection of Inherited
10 DNA Polymorphisms and Human Diseases 344
Identification 260 THE MOLECULAR BASIS OF INHERITED DISEASES 345
TYPES OF POLYMORPHISMS 261 CHROMOSOMAL ABNORMALITIES 345
RFLP TYPING 262 PATTERNS OF INHERITANCE IN SINGLE-GENE DISORDERS 346
Genetic Mapping With RFLPs 264 MOLECULAR BASIS OF SINGLE-GENE DISORDERS 349
RFLP and Parentage Testing 264 Lysosomal Storage Diseases 349
Human Identification Using RFLPs 265 Factor V Leiden 349
STR TYPING BY PCR 266 Prothrombin 349
STR Analysis 268 Methylenetetrahydrofolate Reductase 352
Y-STR 278 Hemochromatosis 352
LINKAGE ANALYSIS 282 Cystic Fibrosis 353
Cytochrome P-450 354
BONE MARROW ENGRAFTMENT TESTING USING DNA
SINGLE-GENE DISORDERS WITH NONCLASSICAL PATTERNS
POLYMORPHISMS 283
OF INHERITANCE 355
PSTR Testing 285
Mutations in Mitochondrial Genes 356
Post-Transplant Engraftment Testing 287
Nucleotide-Repeat Expansion Disorders 357
QUALITY ASSURANCE FOR SURGICAL SECTIONS
Genomic Imprinting 362
USING STR 289
Multifactorial Inheritance 363
SINGLE-NUCLEOTIDE POLYMORPHISMS 289
LIMITATIONS OF MOLECULAR TESTING 363
The Human Haplotype Mapping (HapMap) Project 290
MITOCHONDRIAL DNA POLYMORPHISMS 291 13 Molecular Oncology 369
OTHER IDENTIFICATION METHODS 293 CLASSIFICATION OF NEOPLASMS 370
Protein-Based Identification 293 MOLECULAR BASIS OF CANCER 371
Epigenetic Profiles 294
ANALYTICAL TARGETS OF MOLECULAR TESTING 372
11 Detection and Identification GENE AND CHROMOSOMAL MUTATIONS IN SOLID TUMORS 372
of Microorganisms 301 Human Epidermal Growth Factor Receptor 2, HER2/neu/erb-b2 1
SPECIMEN COLLECTION 302 (17q21.1) 372
SAMPLE PREPARATION 304 Epidermal Growth Factor Receptor, EGFR (7p12) 373
xvi Contents
Kirsten Rat Sarcoma Viral Oncogene Homolog, K-ras (12p12); HLA Test Discrepancies 436
Neuroblastoma ras, N-ras (1p13); and Harvey Rat Sarcoma Coordination of HLA Test Methods 437
Viral Oncogene Homolog, H-ras (11p15) 375 ADDITIONAL RECOGNITION FACTORS 437
Ewing Sarcoma, EWS (22q12) 376 Minor Histocompatibility Antigens 437
Synovial Sarcoma Translocation, Chromosome 18—Synovial Nonconventional MHC Antigens 437
Sarcoma Breakpoint 1 and 2, SYT-SSX1, SYT-SSX2 t(X;18) Killer Cell Immunoglobulin-Like Receptors 437
(p11.2;q11.2) 377 MHC DISEASE ASSOCIATION 438
Paired Box-Forkhead in Rhabdomyosarcoma, PAX3-FKHR, SUMMARY OF LABORATORY TESTING 439
PAX7-FKHR, t(1;13), t(2;13) 378
Tumor Protein 53, TP53 (17p13) 378 15 Quality Assurance and Quality Control
Ataxia Telangiectasia Mutated Gene, ATM (11q22) 379 in the Molecular Laboratory 446
Breast Cancer 1 Gene, BRCA1 (17q21), and Breast Cancer 2 Gene, SPECIMEN HANDLING 447
BRCA2 (13q12) 380 Collection Tubes for Molecular Testing 448
Von Hippel–Lindau Gene, VHL (3p26) 380 Precautions 450
V-myc Avian Myelocytomatosis Viral-Related Oncogene, Holding and Storage Requirements 451
Neuroblastoma-Derived, MYCN or n-myc (2p24) 381 TEST PERFORMANCE 451
V-Ros Avian UR2 Sarcoma Virus Oncogene Homolog 1 (ROS1) Next-Generation Sequencing 456
Proto-Oncogene (6q22.1) and Rearranged During Transfection Calibrators and Method Calibration 456
(RET) Proto-Oncogene (10q11) 381 Controls 457
Anaplastic Lymphoma Receptor Tyrosine Kinase (ALK) QUALITY CONTROL 458
Proto-Oncogene, 2p23.1 382 QUALITY ASSURANCE 458
V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog, INSTRUMENT MAINTENANCE 459
KIT, c-KIT (4q12) 382 Instrument Calibration 463
Other Molecular Abnormalities 382 REAGENTS 463
Microsatellite Instability 382 Reagent Categories 464
Loss of Heterozygosity 385 Chemical Safety 465
Liquid Biopsy 386 Reagent Storage 466
MOLECULAR ANALYSIS OF LEUKEMIA AND LYMPHOMA 387 Reagent Labeling 466
Gene Rearrangements 387 PROFICIENCY TESTING 468
Mutations in Hematological Malignancies 397 DOCUMENTATION OF TEST RESULTS 468
Mutation Spectra 405 Gene Nomenclature 469
Gene Sequencing Results 469
14 DNA-Based Tissue Typing 417 REPORTING RESULTS 469
THE MHC LOCUS 418
HLA POLYMORPHISMS 420 Appendix A Study Question Answers 473
HLA Nomenclature 420
MOLECULAR ANALYSIS OF THE MHC 425
Appendix B Answers to Case Studies 501
Serological Analysis 427 Glossary 505
DNA-Based Typing 430
Combining Typing Results 436 Index 529
107 copies
106 copies
105 copies
100
104 copies
103 copies
102 copies
101 copies
10
Rn
0.1
1 3 5 7 9 21 23 25 27 29 33 35 37 39 41 43 45 47 49
Cycle
COLOR PLATE 1 A plot of the accumulation of polymerase COLOR PLATE 3 Chromosome painting showing a deriva-
chain reaction (PCR) product over 50 cycles of PCR. In this tive chromosome formed by the movement of a fragment of
sigmoid curve, the generation of fluorescence occurs earlier chromosome 12 (black) to an unidentified chromosome. See
with more starting template (solid lines) than with less (dotted Figure 7.19 in the text.
lines). See Figure 6.13A in the text.
Cell nucleus
Probes
COLOR PLATE 4 Multicolor fluorescence in situ hybrid-

ization (FISH) analysis simultaneously reveals structural or
numerical abnormalities in three loci. See Figure 7.21 in the
text.
Translocated
chromosome
Reciprocal
translocation
Translocated product
chromosome
COLOR PLATE 2 Fluorescence in situ hybridization (FISH)

analysis using distinct probes to detect a translocation. A
normal nucleus has two signals from each probe (top). A trans-
location involving the two chromosomes combines the two
probe colors (middle). Dual-fusion probes confirm the presence
of the translocation by also giving a signal from the reciprocal
breakpoint (bottom). See Figure 7.16 in the text.
A
C
G
T
CCTTTTTGAAATAAAGNCCTGCCCNGTATTGCTTTAAACAAGATTT
10 20 30 40
CCTCTATTGTTGGATCATTCGTCACAAAATGATTCTGAATTAGCGTATCGT
60 70 80 90 100
COLOR PLATE 5 Electropherogram showing a dye blob at the beginning of a sequence (nucleotide positions 9 to 15). The
sequence read around this area is not accurate. See Figure 9.10 in the text.
A
C
G
T
GATTCTGAATTAGCTGTATCG NNTTSTGNMATYNKCTKNATCG
COLOR PLATE 6 Examples of good sequence quality (left) and poor sequence quality (right). Note the clean baseline on the
good sequence; that is, only one color peak is present at each nucleotide position. Automatic sequence-reading software will not
accurately call a poor sequence. Compare the text sequences below the two scans. See Figure 9.11 in the text.
A
C
G
T
GCTGGTGGCGTA GCTTGTGGCGTAG CTACGCCACAAGC

G C
COLOR PLATE 7 Sequencing of a heterozygous G to T mutation in exon 12 of the KRAS gene. The normal codon sequence is
GGT (left). The heterozygous mutation (GT; center) is confirmed in the reverse sequence (CA; right). See Figure 9.12 in the text.
A
C
G
T
G T A T G C A G A A A A T C T T A G A G T G T C C C A T C T G G T A A G T C A G C
G T A T G C A G A A A A T C T T A G W G T S T C M Y M T S K K G R W A W S T S M R C
COLOR PLATE 8 The 187 delAG mutation in the BRCA1 gene detected by Sanger sequencing. This heterozygous dinucleotide
deletion is evident in the lower panel where, at the site of the mutation, two sequences are overlaid: the normal sequence and the
normal sequence minus two bases. See Figure 9.13 in the text.
0.08
Fluorescence (FZ/Back–F1)
0.07
BK
0.06
0.05
0.04
JC
0.03
0.02
0.01
0
55 60 65 70 75 80 85
Temperature (°C)
Fluorescence, d(FZ/Back–F1)dt
0.012
0.01
COLOR PLATE 9 Melt-curve analysis of BK and JC viruses. BK BK
JC
and JC are differentiated from one another by differences in the 0.008
Tm* of the probe specific for each viral sequence. Fluorescence 0.006
from double-stranded DNA decreases with increasing temperature 0.004
and DNA denaturation to single strands (top panel). Instrument 0.002
software will present the derivative of the fluorescence (bottom
0
panel) where the melting temperatures (Tm; 67°C to 68°C for BK
–0.002
and 73°C to 74°C for JC) are observed as peaks. See Figure 11.4 60 62 64 66 68 70 72 74 76 78 80
in the text. Temperature (°C)
HAZARDOUS MATERIALS
CLASSIFICATION
HEALTH HAZARD FIRE HAZARD

Flash Point
4 Deadly 4 Below 73°F
3 Extreme Danger 3 Below 100°F
2 Hazardous 2 Below 200°F
1 Slightly Hazardous 1 Above 200°F
0 Normal Material
2
0 Will not burn
COLOR PLATE 10 Biohazard stickers are required for cabi-

nets, refrigerators, or freezers that contain potentially hazard-
ous reagents or patient specimens. See Figure 15.1 in the text.
3 1
SPECIFIC
HAZARD
W REACTIVITY
4 May deteriorate
3 Shock and heat
Oxidizer OXY may deteriorate
Acid ACID 2 Violent chemical
Alkali ALK change
Corrosive COR 1 Unstable if
Use No Water W heated
Radiation 0 Stable
COLOR PLATE 12 National Fire Protection Association

(NFPA) hazard labels have three parts, labeled with numbers
0 to 4, depending on the severity of the hazard, from none
(0) to severe (4). The fourth section has two categories. OXY
indicates a strong oxidizer, which greatly increases the rate of
COLOR PLATE 11 For molecular analysis, blood or bone combustion. The W symbol indicates dangerous reactivity with
marrow specimens collected in ethylenediaminetetraacetic water, which would prohibit the use of water to extinguish a
acid (EDTA; lavender-cap) or acid citrate dextrose (ACD; fire in the presence of this chemical. See Figure 15.17 in the
yellow-cap) tubes are preferred. Heparin (green cap) is used text.
for cytogenetic tests. Immunoassays or mass-spectrome-
try methods may be performed on serum collected in tubes
without coagulant (red-cap tubes). See Figure 15.3 in the text.
RADIATION
COLOR PLATE 13 Rooms, cabinets, and equipment contain-
ing radioactive chemicals are identified with radiation safety
labels. See Figure 15.18 in the text.
Section I
Fundamentals of Molecular
Biology: An Overview
1
Chapter 1
Nucleic Acids and Proteins
Outline Transfer RNA

Other RNAs
DNA RNA POLYMERASES
DNA STRUCTURE OTHER RNA-METABOLIZING ENZYMES
Nucleotides Ribonucleases
Nucleic Acid RNA Helicases
DNA REPLICATION PROTEINS AND THE GENETIC CODE
Polymerases Amino Acids
ENZYMES THAT METABOLIZE DNA Genes
Restriction Enzymes The Genetic Code
DNA Ligase TRANSLATION
Other DNA Metabolizing Enzymes Amino Acid Charging
RECOMBINATION IN SEXUALLY REPRODUCING ORGANISMS Protein Synthesis
RECOMBINATION IN ASEXUAL REPRODUCTION
Conjugation
Transduction
Transformation Objectives
PLASMIDS
RNA 1.1 Diagram the structure of nitrogen bases,
Transcription nucleosides, and nucleotides.
Transcription Initiation 1.2 Describe the nucleic acid structure as a polymer of
Transcription Elongation nucleotides.
Transcription Termination 1.3 Demonstrate how deoxyribonucleic acid (DNA)
TYPES/STRUCTURES OF RNA is replicated such that the order or sequence
Ribosomal RNA of nucleotides is maintained (semiconservative
Messenger RNA replication).
Messenger RNA Processing 1.4 Relate how ribonucleic acid (RNA) is synthesized
Small Nuclear RNA (transcription) compared with DNA
1.5 List and describe types of RNA.
2
Chapter 1 • Nucleic Acids and Proteins 3
1.6 Explain the reaction catalyzed by polymerases that limited specimens. Furthermore, information carried in
results in the phosphodiester backbone of the the order or sequence of the nucleotides that make up
nucleic acid chains. the nucleic acids is the basis for normal and pathological
1.7 Note how the replicative process results in the traits from microorganisms to humans and, as such, pro-
antiparallel nature of complementary strands of vides a powerful means of predictive analysis. Effective
DNA. prevention and treatment of disease will result from the
1.8 List the enzymes that modify DNA and RNA, and analysis of these molecules in the medical laboratory.
state their specific functions.
1.9 Explain mRNA processing, including capping,
polyadenylation, and splicing. DNA
1.10 Illustrate three ways in which DNA can be
transferred between bacterial cells. Deoxyribonucleic acid (DNA) is a macromolecule of
1.11 Define recombination, and sketch how new carbon, nitrogen, oxygen, phosphorous, and hydrogen
combinations of genes are generated in sexual atoms. It is assembled in units of nucleotides that are
and asexual reproduction. composed of a phosphorylated ribose sugar and a nitro-
1.12 Outline the structure and chemical nature of the gen base. There are four nitrogen bases that make up
20 amino acids. the majority of DNA found in all organisms. These are
1.13 Show how the chemistry of the amino acids adenine, cytosine, guanine, and thymine. Nitrogen
affects their chemical characteristics. bases are attached to a deoxyribose sugar, which forms
1.14 Give the definition of a gene. a polymer with the deoxyribose sugars of other nucleo-
1.15 Recount how the genetic code was solved. tides through a phosphodiester bond. Linear assembly
1.16 Describe how amino acids are polymerized into of the nucleotides makes up one strand of DNA. Two
proteins, using RNA as a guide (translation). strands of DNA comprise the DNA double helix.
1.17 Relate protein function to the structural domains In 1871, Johann Friedrich Miescher published a
of the amino acid sequence. paper on nuclein, the viscous substance extracted from
cell nuclei. In his writings, he made no mention of the
function of nuclein. Walther Flemming, a leading cell
biologist, describing his work on the nucleus in 1882
When James Watson coined the term molecular biology,1 admitted that the biological significance of the substance
he was referring to the biology of deoxyribonucleic acid was unknown. We now know that the purpose of DNA,
(DNA). Of course, there are other molecules in nature. contained in the nucleus of the cell, is to store informa-
The term, however, is still used to describe the study of tion. The information in the DNA storage system is based
nucleic acids. In the medical laboratory, molecular tech- on the order or sequence of nucleotides in the nucleic
niques are designed for the handling and analysis of the acid polymer. Just as computer information storage is
nucleic acids, DNA and ribonucleic acid (RNA). Protein based on sequences of 0 and 1, biological information is
analysis and that of carbohydrates and other molecular based on sequences of A, C, G, and T. These four build-
species might also be categorized as “molecular” studies ing blocks (with a few modifications) account for all of
performed by flow cytometry, in situ histology, and the biological diversity that makes up life on earth.
tissue typing. The molecular biology laboratory, there-
fore, may be a separate entity or part of an existing clin-
ical pathology unit. This chapter will address the nucleic Histooricaal Higghlligghtts
acids.
Nucleic acids offer several characteristics that support Johann Friedrich Miescher is credited with the
their use for clinical purposes. Highly specific analyses discovery of DNA in 1869.2 Miescher had iso-
can be carried out without the requirement for exten- lated white blood cells out of seepage collected
sive physical or chemical selection of target molecules from discarded surgical bandages. He found that
or organisms, allowing specific and rapid analysis from he could extract a viscous substance from the cells
4 Section I • Fundamentals of Molecular Biology: An Overview
in this material. Miescher also observed that most nucleosides. If the ribose sugar is phosphorylated, the
of the nonnuclear cell components could be lysed molecule is a nucleoside mono-, di-, or triphosphate or a
away with dilute hydrochloric acid, leaving the nucleotide. For example, adenosine with one phosphate
nuclei intact. Addition of extract of pig stomach is adenosine monophosphate (AMP). Adenosine with
(a source of pepsin to dissolve away contaminat- three phosphates is adenosine triphosphate (ATP). Free
ing proteins) resulted in a somewhat shrunken but nucleotides are deoxyribonucleoside triphosphates (e.g.,
clean preparation of nuclei. Extraction of these dATP). They are routinely designated as A, C, G, and T
with alkali yielded the same substance isolated in the DNA molecule. Nucleotides can be converted to
from the intact cells. It precipitated upon the addi- nucleosides by hydrolysis.
tion of acid and redissolved in alkali. Chemical The five-carbon sugar of DNA is deoxyribose, which
analysis of this substance demonstrated that it is ribose with the number-two carbon of deoxyribose
was 14% nitrogen and 2.5% phosphorus, different linked to a hydrogen atom rather than a hydroxyl group
from any then-known group of biochemicals. He (see Fig. 1.2). The hydroxyl group on the third carbon
named the substance “nuclein.” (Analytical data is important for forming the phosphodiester bond that is
indicate that less than 30% of Miescher ’s first the backbone of the DNA strand.
nuclein preparation was actually DNA.) He later Nitrogen bases are planar carbon-nitrogen ring struc-
isolated a similar viscous material from salmon tures. The four common nitrogen bases in DNA are
sperm and noted: “If one wants to assume that a adenine, guanine, cytosine, and thymine. Amine and
single substance . . . is the specific cause of fertil- ketone substitutions, as well as the single or double
ization, then one should undoubtedly first of all bonds within the rings, distinguish the four bases that
think of nuclein.” comprise the majority of DNA (Fig. 1.3). Nitrogen
bases with a single-ring structure (thymine, cytosine)
are pyrimidines. Bases with a double-ring structure
(guanine, adenine) are purines.
The numbering of the positions in the nucleotide
DNA STRUCTURE molecule starts with the ring positions of the nitrogen
base, designated C or N 1, 2, 3, and so on. The carbons
The double helical structure of DNA (Fig. 1.1) was first of the ribose sugar are numbered 1′ to 5′, distinguishing
described by James Watson and Francis Crick. Their the positions of the sugar rings from those of the nitro-
molecular model was founded on previous observations gen base rings (Fig. 1.4).
of the chemical nature of DNA and physical evidence
including diffraction analyses performed by Rosalind
Franklin.3 The helical structure of DNA results from the
physicochemical demands of the linear array of nucleo-
tides. Both the specific sequence (order) of nucleotides Advanced Concepts
in the strand, as well as the surrounding chemical micro-
environment, can affect the nature of the DNA helix. The double helix first described by Watson and
Crick is DNA in its hydrated form (B-form) and
is the standard form of DNA.4 It has 10.5 steps
Nucleotides
or pairs of nucleotides (base pairs [bp]) per turn.
The four nucleotide building blocks of DNA are mol- Dehydrated DNA takes the A-form with about
ecules of about 700 kd. Each nucleotide consists of a 11 bp per turn and the center of symmetry along
five-carbon sugar, the first carbon of which is covalently the outside of the helix rather than down the
joined to a nitrogen base and the fifth carbon to a phos- middle as it is in the B-form. Both A- and B-form
phate moiety (Fig. 1.2). A nitrogen base bound to an DNA are right-handed helices. Stress and torsion
unphosphorylated sugar is a nucleoside. Adenosine (A), can throw the double helix into a Z-form. Z-DNA
guanosine (G), cytidine (C), and thymidine (T) are
Nitrogen bases
3′ 5′
C C G C C G C
G T A G
DNA A A G A
A A
double helix C C T G
T C
G C G
C C
5′ G C A T G
3′
C
T T G C G T
A Sugar-phosphate backbone
Hydrogen bond
H
CH3 O H N N
CH
C C C C
A N
HC T N H N C
N C HC N
O
Thymine Adenine
H
N H O N
CH
HC C C C
G N
HC C N H N C
N C C N
O H NH
B Cytosine Guanine
FIGURE 1.1 (A) The double helix. The phosphodiester backbones of the two nucleic acid chains form the helix. Nitrogen bases
are oriented toward the center, where they hydrogen bond with homologous bases to stabilize the structure. (B) Two hydrogen
bonds form between adenine and thymine. Three hydrogen bonds form between guanine and cytosine.
Phosphate N C
group OR Guanine
HC C NH
O– P O
N C C
O O N NH2
FIGURE 1.2 The nucleotide deoxyguanosine 5′ phosphate or 1
H2C CH CH
guanosine monophosphate (dGMP). It is composed of deoxyri- 5 Ribose
bose covalently bound at its number 1 carbon to the nitrogen HC CH2
base, guanine, and at its number 5 carbon to a phosphate group.
The molecule without the phosphate group is the nucleoside, OH
deoxyguanosine. dGMP
PURINES
O NH2
Guanine N C Adenine N C
OR OR
HC C NH HC C N
O– P O O– P O
N C C N C HC
O O N NH2 O O N
H2C CH CH2 H 2C CH CH2
HC CH2 HC CH2
OH OH
dGMP dAMP
PYRIMIDINES
NH2 O
Cytosine C Thymine C
HC N H3C C NH
OR OR
O– P O HC C O O– P O HC C O
N N
O O O O
H2C CH CH H2C CH CH
HC CH2 HC CH2
OH OH
dCMP dTMP
FIGURE 1.3 Nucleotides, deoxyguanosine monophosphate (dGMP), deoxyadenosine monophosphate (dAMP), deoxythymidine
monophosphate (dTMP), and deoxycytidine monophosphate (dCMP), differ by the attached nitrogen bases. The nitrogen bases,
guanine and adenine, have purine ring structures. Thymine and cytosine have pyrimidine ring structures. Uracil, the nucleotide
base that replaces thymine in RNA, has the pyrimidine ring structure of thymine minus the methyl group and hydrogen bonds with
adenine.
O
7 6
8 N 5 C 1
OR
HC C NH
O– P O
9 N C C2
O O 4 N NH2
3 FIGURE 1.4 Carbon position numbering of a nucleotide monophos-
H2C CH CH
1′ phate. The base carbons are numbered 1 through 9. The sugar carbons
5′ 4′
HC CH2
are numbered 1′ to 5′. The phosphate group on the 5′ carbon and the
3′ 2′ hydroxyl group on the 3′ carbon form phosphodiester bonds between
OH bases. The 1′ carbon holds the nitrogen base.
is a left-handed helix with 12 bp per turn and Advanced Concepts

altered geometry of the sugar-base bonds. Z-DNA
has been observed in areas of chromosomes where In addition to the four commonly occurring nucle-
the DNA is under torsional stress from unwinding otide bases, modified bases are also often found in
for transcription or other metabolic functions. nature. Base modifications have significant effects
Watson–Crick base pairing (purine:pyrimidine on phenotype. Some modified bases result from
hydrogen bonding) is not limited to the ribofu- damage to DNA; others are naturally modified for
ranosyl nucleic acids, those found in our genetic specific functions or to affect gene expression, as
system. Natural nucleic acid alternatives can also will be discussed in later sections.
display the basic chemical properties of DNA (and Modified nucleotides are used by bacteria and
RNA). Theoretical studies have addressed such viruses as a primitive immune system that allows
chemical alternatives to DNA and RNA compo- them to distinguish their own DNA from that of
nents. An example is the pentopyranosyl-(2′→4′) the host or invaders (restriction-modification [RM]
oligonucleotide system that exhibits stronger and system). Recognizing its own modifications, the
more selective base pairing than DNA or RNA.5 host can target unmodified DNA for degradation.
The study of nucleic acid alternatives has practical
applications. For example, protein nucleic acids,
which have a carbon-nitrogen peptide backbone Due to their effects on enzymes that metabolize DNA,
replacing the sugar-phosphate backbone,6,7 can be modified nucleosides have been used effectively for
used in the laboratory as alternatives to DNA and clinical applications (Fig. 1.5). The anticancer drugs
RNA hybridization probes.8,9 They have also been 5-bromouridine (5BrdU) and cytosine arabinoside
proposed as potential enzyme-resistant alternatives (cytarabine, ara-C) are modified thymidine and cytosine
to RNA in antisense RNA therapies.10
Nitrogen base Nitrogen base
HOCH2 O HOCH2 O
C C C C
Two DNA chains form hydrogen bonds with each other C C C
C
in a specific way. Guanines in one chain form three
OH
hydrogen bonds with cytosines in the opposite chain,
Deoxynucleoside Dideoxynucleoside
and adenines form two hydrogen bonds with thymines
(see Fig. 1.1B). In this way, single nucleic acid strands
can bind or hybridize to single strands that have the cor-
responding bases. Hydrogen bonds between nucleotides HOCH2 O T HOCH2 O G
are the key to the specificity of most nucleic acid–based
C C C C
tests used in the molecular laboratory. Specific hydrogen
bond formation is also how the information held in the C C C C
linear order of the nucleotides is maintained. As DNA N3 OH
is polymerized, each nucleotide to be added to the new Azidothymidine Acyclovir
DNA strand hydrogen bonds with the complementary (AZT)
nucleotide on the parental strand (A:T, G:C). In this way FIGURE 1.5 Substituted nucleosides used in the clinic and
the parental DNA strand can be replicated without loss the laboratory. Dideoxynucleosides are used as laboratory
of the nucleotide order. Base pairs other than A:T and reagents. Azidothymidine is an antiviral drug that inhibits the
G:C or mismatches (e.g., A:C, G:T, A:A) can distort human immunodeficiency virus and is used to treat AIDS.
the DNA helix and disrupt the maintenance of sequence Another antiviral, acyclovir, inhibits the growth of herpes
information. viruses.
nucleosides, respectively. Azidothymidine (Retrovir, Growing strand

AZT), cytosine, 2′,3′-dideoxy-2′-fluoro (ddC), and 2′,3′-
dideoxyinosine (Videx, ddI), drugs used to treat patients
with human immunodeficiency virus (HIV) infections, O– P O
are modifications of thymidine and cytosine and a pre- O
Template strand
cursor of adenine, respectively. An analog of guanosine,
H2C O
2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]- A T
6H-purin-6-one (Acyclovir, Zovirax) is a drug used to CH CH2
combat herpes simplex virus and varicella-zoster virus.
HC CH2
In the laboratory, nucleosides can be modified for
the purposes of labeling or detecting DNA molecules, O
sequencing, and other applications. The techniques used O– P O
for these procedures will be discussed in later chapters.
O
H2C O
G C
Nucleic Acid
CH CH2
Nucleic acid is a macromolecule made of nucleotides
bound together by the phosphate and hydroxyl groups HC CH2
on their sugars. A nucleic acid chain grows by the attach- OH

ment of the 5′ phosphate group of an incoming nucleo- Phosphodiester
tide to the 3′ hydroxyl group of the last nucleotide on the linkage
growing chain (Fig. 1.6). Addition of nucleotides in this

way gives the DNA chain a polarity; that is, it has a 5′ O O O
phosphate end and a 3′ hydroxyl end. We refer to DNA O– P O P O P O–

as oriented in a 5′ to 3′ direction, and the linear sequence
O– O– O C G
of the nucleotides, by convention, is read in that order.
H2C O
Pyrophosphate
CH CH
HC CH2
OH
Advanced Concepts
Incoming nucleotide
The sugar-phosphate backbones are arranged at
specific distances from one another in the double FIGURE 1.6 DNA replication is a template-guided poly-
helix (see Fig. 1.1). The two regions formed in merization catalyzed by DNA polymerase. The new strand is
the helix by the backbones are called the major synthesized in the 5′ to 3′ direction, reading the template strand
groove and minor groove. The major and minor in the 3′ to 5′ direction.
grooves are sites of interaction with the many pro-
teins that bind to specific nucleotide sequences in
DNA (binding or recognition sites). The double DNA found in nature is mostly double stranded. Two
helix can also be penetrated by intercalating strands exist in opposite 5′ to 3′/3′ to 5′ orientation, held
agents, molecules that slide transversely into the together by the hydrogen bonds between their respective
center of the helix. Denaturing agents such as bases (A with T and G with C). The bases are positioned
formamide and urea displace the hydrogen bonds such that the sugar-phosphate chain that connects them
and separate the two strands of the helix. (sugar-phosphate backbone) is oriented in a spiral or
helix around the nitrogen bases (see Fig. 1.1).
The DNA double helix represents two versions of the DNA REPLICATION
information stored in the form of the order or sequence
of the nucleotides on each chain. The sequences of the The two DNA strands of a double helix have an antipar-
two strands that form the double helix are complemen- allel orientation because of the way DNA is replicated.
tary, not identical (Fig. 1.7). They are in antiparallel As DNA synthesis proceeds in the 5′ to 3′ direction,
orientation, with the 5′ end of one strand at the 3′ end DNA polymerase, the enzyme responsible for polymer-
of the other (Fig. 1.8). The formation of hydrogen bonds izing the nucleotide chains, uses a guide, or template,
between two complementary strands of DNA is called to determine which nucleotides to add to the chain. The
hybridization. Single strands of DNA with identical enzyme reads the template in the 3′ to 5′ direction. The
sequences will not hybridize with each other. Later sec- resulting double strand, then, will have a parent strand in
tions will describe the importance of this when design- one orientation and a newly synthesized strand arranged
ing assays. in the opposite orientation.
As Watson and Crick predicted, semi-conservative
PO 5′ G T A G C T C G C T G A T 3′ OH replication is the key to maintaining the sequence of
HO 3′ C A T C G A G C G A C T A 5′ OP the nucleotides in DNA through new generations. It is
important that this information, in the form of the DNA
FIGURE 1.7 Homologous sequences are not identical and are sequence, be transferred faithfully at cell division. The
oriented in opposite directions. replication apparatus is designed to copy the DNA
strands in an orderly way with minimal errors before
each cell division.
5′ 3′
OH
Histooricaal Higghlligghtts
A T Before the double helix was determined, Erwin
Chargaff11 made the observation that the amount
of adenine in DNA corresponded to the amount of
thymine and the amount of cytosine to the amount
of guanine. Upon the description of the double
G C helix, Watson proposed that the steps in the ladder
of the double helix were pairs of bases, thymine
with adenine and guanine with cytosine. Watson
and Crick, upon publication of their work, sug-
gested that this arrangement was the basis for a
copying mechanism. The complementary strands
C G could separate and serve as guides or templates
for producing complementary strands.
3′ 5′
HO In the process of replication, DNA is first unwound

from the helical duplex so that each single strand may
serve as a template for the addition of nucleotides to the
FIGURE 1.8 Because DNA synthesis proceeds from the 5′ new strand (see Fig. 1.6). The new strand is elongated
phosphate group to the 3′ hydroxyl group and the template by hydrogen bonding of the complementary incoming
strand is copied in the opposite (3′ to 5′) direction, the new nucleotide to the nitrogen base on the template strand and
double helix consists of the template strand and the new daugh- then a nucleophilic attack of the deoxyribose 3′ hydroxyl
ter strand oriented in opposite directions from one another. oxygen on a phosphorous atom of the phosphate group
on the hydrogen-bonded nucleotide triphosphate. Ortho- by electron microscopy as a forked structure, or repli-
phosphate is released with the formation of a phospho- cation fork. Note, however, that the antiparallel nature
diester bond between the new nucleotide and the last of duplex DNA and the requirement for the DNA syn-
nucleotide of the growing chain. The duplicated helix thesis apparatus to read the template strand in a 3′ to
will ultimately consist of one template strand and one 5′ direction are not consistent with copying of both
newly synthesized strand. strands simultaneously in the same direction. The ques-
tion arises as to how one of the strands of the duplex
Histooricaal Higghlligghtts can be copied in the same direction as its complemen-
tary strand that runs antiparallel to it. This problem
A few years after the solution of the double helix, was addressed in 1968 by Okazaki and Okazaki13 when
the mechanism of semiconservative replication studying DNA replication in Escherichia coli. In their
was demonstrated by Matthew Meselson and experiments, small pieces of DNA, about 1,000 bases
Franklin Stahl,12 using the technique of equilib- in length, could be observed by density-gradient cen-
rium density centrifugation on a cesium gradient. trifugation in actively replicating DNA. The fragments
They prepared “heavy” DNA by growing bacte- changed into larger pieces with time, showing that they
ria in a medium containing the nitrogen isotope were covalently linked together shortly after synthesis.
15
N. After shifting the bacteria into a medium of These small fragments, or Okazaki fragments, were
normal nitrogen (14N), they could separate hybrid the key to explaining how both strands were copied
14
N:15N DNA molecules synthesized as the bacte- at the replication fork. The two strands of the parent
ria replicated. These molecules were of a specific helix are not copied in the same way. While DNA rep-
intermediate density to the ones from bacteria lication proceeds in a continuous manner on the 3′ to
grown only in 14N or 15N. They could differentiate 5′ strand, or the leading strand, the replication apparatus
true semiconservative replication from dispersive jumps ahead a short distance (~1,000 bases) on the 5′ to
replication by demonstrating that approximately 3′ strand and then copies backward toward the replica-
half of the DNA double helices from the next tion fork. The 5′ to 3′ strand copied in a discontinuous
generation grown in 14N were 14N:15N, and half manner is the lagging strand14 (Fig. 1.9).
were 14N:14N. Another requirement for DNA synthesis is the avail-
ability of a deoxyribose 3′-hydroxyl oxygen for chain
growth. This means that DNA cannot be synthesized de
DNA replication proceeds through the DNA duplex novo; a preceding base must be present to provide the
with both strands of DNA replicating in a single pass. hydroxyl group. This base is provided by another enzyme
DNA undergoing active replication can be observed component of the replication apparatus, primase.15
DNA
polymerase
3′
5′ 5′
Leading strand 3′
Lagging strand 5′
3′ 3′
Replication fork
5′
Okazaki fragments
Overall direction of replication
FIGURE 1.9 Simultaneous replication of both strands of the double helix. Both strands are read in the 3′ to 5′ direction. One
strand oriented 5′ to 3′ (the lagging strand) is read discontinuously, with the polymerase skipping ahead and reading back toward
the replication fork.
Primase is an RNA-synthesizing enzyme that catalyzes

the synthesis of short (6 to 11 bp) RNA primers required TABLE 1.1 Examples of Polymerases Classified
for priming DNA synthesis. Primase must work repeat- by Sequence Homology
edly on the lagging strand to prime synthesis of each
Okazaki fragment. Family Polymerase Source Activity
A Pol I E. coli Recombination,

repair,
Advanced Concepts replication
A T5 pol, T7 pol T5, T7 Replication

The DNA replication complex (replisome) contains bacteriophage
all the necessary proteins for the several activities
involved in faithful replication of double-stranded A Pol γ Mitochondria Replication
DNA. Helicase activity in the replisome unwinds
B Pol II E. coli Repair
and untangles the DNA for replication. Primase
functions either as a separate protein or in a B Archael P. furiosus Replication,
primase-helicase polyprotein in the replisome. repair
The E. coli primase, DnaG, transcribes 2,000 to B φ29 pol, T4 φ29, T4 Replication
3,000 RNA primers at a rate of 1 per second in the pol bacteriophage
replication of the E. coli genome. Separate poly-
merase proteins add incoming nucleotides to the B Polα, PolΔ, Eukaryotes Repair
Polε
growing DNA strands of the replication fork. The
details of the synthesis of the lagging strand are not B Viral pols Various viruses Repair
yet clear, although recent evidence suggests that
C Pol III core E. coli Replication
discontinuous replication proceeds by a ratcheting
mechanism, with replisome molecules pulling the C dnaE, dnaEBS B. subtilis Replication
lagging strand in for priming and copying.16 Once
X Pol β Eukaryotes Repair,
DNA is primed and synthesized, RNase H, an
replication
enzyme that hydrolyzes RNA from a complemen-
tary DNA strand, removes the primer RNA from A Polη, Polι Eukaryotes Bypass
the short RNA–DNA hybrid, and the resulting gap replication
is filled by gap-filling DNA polymerase. Y Polκ Eukaryotes Bypass
replication,
cohesion
Polymerases Y Pol IV, Pol V E. coli Bypass

replication
The first purified enzyme shown to catalyze DNA repli-
cation in prokaryotes was designated DNA polymerase I Y Rev1, Rad30 S. cerevisiae Bypass
replication
(pol I). DNA polymerases II (pol II) and III were later
UV-induced
characterized, and it was discovered that DNA poly- repair
merase III (pol III) was the main polymerizing enzyme
during bacterial replication (Table 1.1). The other two B Rad 6, Polξ S. cerevisiae
polymerases were found to be responsible for the repair
of gaps and discontinuities in previously synthesized In vivo, pol III functions as a multi-subunit holo-
DNA. It is not surprising that pol I was preferentially enzyme. The holoenzyme works along with a larger
purified in those early studies. In in vitro studies where assembly of proteins required for priming, initiation,
the enzymes were first described, pol II and pol III regulation, and termination of the replication process
activity was less than 5% of that of pol I. (Fig. 1.10). Two of the 10 subunits of the holoenzyme
Leading strand DNA

polymerase
5′ Single-strand
binding proteins
3′ 5′
Lagging strand
Helicase
5′ 3′
3′
Primase
RNA primer
FIGURE 1.10 DNA polymerase activity involves more than one protein molecule. Several cofactors and accessory proteins are
required to unwind the template helix, prime synthesis with RNA primers, and protect the lagging strand.
are catalytic DNA polymerizing enzymes, one for the new DNA was a copy of the input molecule or
synthesis of the leading strand and one for the synthesis an extension of it. During the next 3 years, Julius
of the lagging strand.16 Adler, Sylvy Kornberg, and Steven B. Zimmer-
man showed that the new DNA had the same
ratio of A-T to G-C bp as the input DNA and was
Advanced Concepts indeed a copy of it. This ratio was not affected
by the proportion of free nucleotides added to the
Genome sequencing has revealed that the organi- initial reaction, confirming that the input or tem-
zation of the proteins in and associated with the plate DNA determined the sequence of the nucle-
holoenzyme is conserved across genera.17 The otides on the newly synthesized DNA.
conserved nature of the polymerase complex sug-
gests a limited range of possible structures with
Most DNA polymerases have more than one function,
polymerase activity. It also explains how a bacte-
including, in addition to polymerization, pyrophos-
rial polymerase can replicate DNA from diverse
phorolysis and pyrophosphate exchange, the latter two
sources. This is important in the laboratory where
activities being a reversal of the polymerization process.
prokaryote polymerases are used extensively to
DNA polymerase enzymes thus have the capacity to
copy DNA from many different organisms.
synthesize DNA in a 5′ to 3′ direction and degrade DNA
in both a 5′ to 3′ and 3′ to 5′ direction (Fig 1.11). The
catalytic domain of E. coli DNA pol I has two frag-
Histooricaal Higghlligghtts ments carrying the two functions, a large fragment with
the polymerase activity and a small fragment with the
At a conference on the chemical basis of heredity exonuclease activity. The large fragment without the
held at Johns Hopkins University in June 1956, exonuclease activity (Klenow fragment) has been used
Arthur Kornberg, I. Robert Lehman, and Maurice extensively in the laboratory for in vitro DNA synthesis.
J. Bessman reported on an extract of E. coli that One purpose of the exonuclease function in the
could polymerize nucleotides into DNA in vitro.14 various DNA polymerases is to protect the sequence of
It was noted that the reaction required preformed nucleotides, which must be faithfully copied. Copying
DNA and all four nucleotides along with a bac- errors will result in base changes or mutations in the
terial protein extract. Any source of preformed DNA. The 3′ to 5′ exonuclease function is required to
DNA would work, bacterial, viral, or animal. At ensure that replication begins or continues with a cor-
the time, it was difficult to determine whether the rectly base-paired nucleotide. The enzyme will remove
3′ 3′
G G
C C
T T
A A
T T
5′ 5′
3′–5′ exonuclease
T T C
A A
3′ C 3′
A A
T T
A A
G G
C C
5′ 5′
Mispair (AC) at 3′ end of Mispaired base (C) removed
growing DNA strand by exonuclease. DNA polymerase
tries a second time.
FIGURE 1.11 DNA polymerase can remove misincorporated bases during replication using its 3′ to 5′ exonuclease activity.
a mismatch (e.g., A opposite C instead of T on the tem- 5′ 3′

plate) in the primer sequence before beginning poly- 3′ 5′
merization. During DNA synthesis, this exonuclease
function gives the enzyme the capacity to proofread Nick
newly synthesized DNA, that is, to remove a misincor- 5′ 3′ 5′ 3′
3′ 5′
porated nucleotide by breaking the phosphodiester bond
and replace it with the correct one.
DNA polymerase
During DNA replication, E. coli DNA pol III can 5′ to 3′ synthesis
synthesize and degrade DNA simultaneously. At a nick, 5′ 3′
3′ 5′
or discontinuity, in one strand of a DNA duplex, the
enzyme can add nucleotides at the 3′ end of the nick
while removing nucleotides ahead of it with its 5′ to DNA ligase
3′ exonuclease function (Fig. 1.12). This concurrent syn- 5′
Newly synthesized DNA
3′
thesis and hydrolysis then moves the nick in one strand 3′ 5′
of the DNA forward in an activity called nick transla-
tion. The polymerization and hydrolysis will proceed for Closed nick
a short distance until the polymerase is dislodged. The 5′ 3′
nick can then be re-closed by DNA ligase, an enzyme 3′ 5′
that forms phosphodiester bonds between existing DNA
FIGURE 1.12 Nick translation of DNA. DNA polymerase
strands. Nick translation is often used in vitro as a
extends the 3+ end of a nick in double-stranded DNA with a
method to introduce labeled nucleotides into DNA mol- newly synthesized strand (gray) while digesting the original
ecules. The resulting labeled products are used for DNA strand from the 5+ end. After polymerization, the nick is closed
detection in hybridization analyses. by DNA ligase.
Advanced Concepts
except they have less demonstrable exonuclease
Like prokaryotes, eukaryotic cells contain multi- activity. A fourth polymerase, δ, originally iso-
ple polymerase activities. Two polymerase protein lated from bone marrow, has 3′ to 5′ exonuclease
complexes, designated α and β, are found in activity. Polymerase α, the most active, is identi-
the nucleus and one, γ, in the mitochondria. The fied with chromosome replication, and β and δ are
three polymerases resemble prokaryotic enzymes, associated with DNA repair.
(faithful copying of the template), and substrate speci-

Advanced Concepts ficity (affinity for altered nucleotides).20
After replication, distortions in the DNA duplex

caused by mismatched or aberrantly modified ENZYMES THAT METABOLIZE DNA
bases are removed by the 5′ to 3′ exonuclease
function of repair polymerases such as DNA pol Once DNA is polymerized, it is not static. The infor-
I. This activity degrades duplex DNA from the mation stored in the DNA must be tapped selectively
5′ end and can also cleave diester bonds several to make RNA and, at the same time, protected from
bases from the end of the chain. It is important damage. Protective systems in prokaryotes eliminate
for removing lesions in the DNA duplex such as foreign DNA, such as infecting bacteriophages or other
thymine or pyrimidine dimers, boxy structures viruses. Enzymes that modify and digest foreign DNA
formed between adjacent thymines or cytosines prevent infection. In sexually reproducing organisms,
and thymines on the same DNA strand that are the mixing of parental DNA generates genetic diversity
induced by exposure of DNA to ultraviolet light. If (hybrid vigor) in the offspring. This process requires
these structures are not removed, they can disrupt cutting and reassembly of the DNA strands in advance of
subsequent transcription and replication of the cell division and gamete formation. A host of enzymes
DNA strand. performs these and other functions during various stages
of the cell cycle. Some of these enzymes, including
DNA polymerase, have been isolated for in vitro manip-
ulation of DNA in the laboratory. They are key tools of
One type of DNA polymerase, terminal transferase, recombinant DNA technology, the basis for commonly
can synthesize polynucleotide chains without a template. used molecular techniques.21
This enzyme will add nucleotides to the end of a DNA
strand in the absence of hydrogen base pairing with a
Restriction Enzymes
template. The initial synthesis of a large dA-dT polymer
by terminal transferase was a significant event in the Genetic engineering was stimulated by the discovery of
history of DNA polymerase studies.18 Terminal transfer- deoxyriboendonucleases, or endonucleases. Endonucle-
ase is used in the laboratory to generate 3′-end labeled ases break the sugar-phosphate backbone of DNA.
DNA species.
DNA polymerases play a central role in modern
biotechnology. Cloning and some amplification and Advanced Concepts
sequencing technologies require DNA polymerase activ-
ity. The prerequisite for specific polymerase characteris- There are several types of endonucleases. Some pre-
tics has stimulated the search for new polymerases and fer single-stranded DNA, and some prefer double-
the engineering of available polymerase enzymes. Poly- stranded DNA. Repair endonucleases function at
merases from various sources are classified into families areas of distortion in the DNA duplex, such as
(A, B, C, X) based on sequence structure.19,20 A short baseless (apurinic or apyrimidic) sites on the DNA
summary is shown in Table 1.1. Other classifications are backbone, thymine dimers, or mismatched bases.
based on structural similarities. Polymerases in the A and Because the chemical structure of DNA is the
B family are most useful for biotechnological engineer- same in all organisms, most enzymes are active on
ing because the polymerase activity of these enzymes is DNA from diverse sources.
contained in a monomeric or single protein. Chemical
manipulation of the amino acid structure produces poly-
merases with characteristics that are useful in the labo- Restriction enzymes are endonucleases that recog-
ratory. These include altered processivity (staying with nize specific base sequences and break or restrict the
the template longer to make longer products), fidelity DNA polymer at the sugar-phosphate backbone. These
TABLE 1.2 Types of Restriction Enzymes
Type Methylation Activity Binding Site Example Cut Site Example
I + AMeCN6G T Variable distance from binding EcoKI, CfrAI
II – GAATTC Within binding EcoR1, BglII

GCCN5GGC
IIS – ACCTGC 1–20 bases from binding BfuAI
IIG + CTGAAG To one side or both sides of binding AcuI

CGAN6TGC BglI
III + CGAAT 25–27 bp 3′ to binding HinfIII

CTGATG (hemimethylated) PstIII
IV + GGGAC Asymetrical Bsplu11III

GMeCCGC SauUSI
enzymes were originally isolated from bacteria, where ability to both methylate and restrict (cut) DNA. Like
they function as part of a primitive immune system to type I, they have multiple subunits, including heli-
cleave foreign DNA entering the bacterial cell. The case (unwinding) activity.22 Recognition sites for these
ability of the cell to recognize foreign DNA depends on enzymes are asymmetrical, and the cleavage of the
both DNA sequence recognition and DNA methylation. substrate DNA occurs 24 to 26 bp from the site to the
Restriction enzymes are named for the organism from 3′ side. An example of a type III enzyme is PstIIII from
which they were isolated. For example, BamHI was iso- P. stuartii.22,23 It recognizes the following site:
lated from Bacillus amyloliquefaciens H, HindIII from
Haemophilus influenzae Rd, SmaI from Serratia marc- 5′ C T G A T G
escens Sbb, and so forth. G A C T A Me C 5′
Restriction endonucleases have been classified into where the adenine methylation occurs on only one
four general types (Table 1.2). Type I restriction enzymes strand. The enzyme cuts the DNA 25 to 26 bp 3′ to the
have both nuclease and methylase activity in a single recognition site.
enzyme. They bind to host-specific DNA sites of 4 to Type IV restriction enzymes have similar subunit
6 bp separated by 6 to 8 bp and containing methylated structures and enzyme requirements. Type IV enzymes
adenines. The site of cleavage of the DNA substrate can have cutting and methyltransferase functions. An example
be over 1,000 bp from this binding site. An example of a of type IV restriction enzymes is BseMII from Bacillus
type I enzyme is EcoK from E. coli K 12. It recognizes stearothermophilus. The BseMII target sequence is
the following site:
5′ C T C A G
5′-A Me C N N N N N N G T G C G A G T C 5′
T G N N N N N N C A Me C G-5′
The restriction endonuclease function of BseMII cuts
where N represents nonspecific nucleotides, and the one strand of DNA 10 bp following the recognition
adenine residues are methylated (AMe). sequence. BseMII also has a methylation function,
Although not completely characterized, type III adding methyl groups to both of the adenine residues in
restriction enzymes resemble type I enzymes in their the target sequence.24
5′ G A A T T C 3′ 5′ C C C G G G 3′ 5′ C T G C A G 3′
DNA
3′ C T T A A G 5′ 3′ G G G C C C 5′ 3′ G A C G T C 5′
Eco RI SmaI Pst l

5′ overhang blunt 3′ overhang
FIGURE 1.13 Type II restriction enzymes recognize symmetrical DNA sequences and cut the sugar-phosphate backbone in dif-
ferent ways, leaving no single strands at the cut site (blunt ends) or 5′ or 3′ overhanging single-stranded ends. Exposed
single-stranded ends are “sticky” ends that can hybridize with complementary overhangs.
Type II restriction enzymes are those used most fre-

quently in the laboratory. These enzymes do not have Advanced Concepts
inherent methylation activity. They bind as simple
dimers to symmetrical 4- to 8-bp DNA recognition sites. Type II enzymes are subclassified by their charac-
These sites are palindromic in nature; that is, they read teristic activities. Type IIS restriction endonucle-
the same 5′ to 3′ on both strands of the DNA (Fig. 1.13), ases (shifted cleavage, e.g., Fok I, Alw1) recognize
referred to as bilateral symmetry. Type II restriction 5- to 7-bp nonpalindromic sequences and cut DNA
enzymes cleave the DNA directly at the binding site, within 20 bases of the recognition site. Type IIT
producing fragments of predictable size. enzymes are composed of two subunits, each of
Type II restriction enzymes have been found in which contains one catalytic site. Type IIM restric-
almost all prokaryotes, but none, to date, have been tion enzymes (e.g., Dpn1) cut methylated DNA.
found in eukaryotes. The specificity of their action Methylation-specific enzymes are a useful tool in
and the hundreds of enzymes available that recognize detecting methylation in DNA.
numerous sites are key factors in the ability to perform
DNA recombination in vitro. Cutting DNA at specific
sequences is the basis of many procedures in molecular
technology, including mapping, cloning, genetic engi- The advantage of blunt ends for in vitro recombination
neering, and mutation analysis. Restriction enzymes are is that blunt ends formed by different enzymes can be
frequently used in the medical laboratory, for example, joined, regardless of the recognition site. This is not true
in the analysis of gene rearrangements and in mutation for sticky ends, which must have matching overhangs.
detection. Sticky ends can be converted to blunt ends using DNA
Although all type II restriction enzymes work with polymerase to extend the recessed strand in a sticky end,
bilateral symmetry, their patterns of double-stranded using the nucleotides of the overhang as a template or
breaks differ (see Fig. 1.13). Some enzymes cut the by using a single-strand exonuclease to remove the over-
duplex with a staggered separation at the recognition hanging nucleotides. Synthetic short DNA fragments
site, leaving 2- to 4-base single-strand overhangs at the with one blunt end and one sticky end (adaptors) can be
ends of the DNA. The single-strand ends can hybridize used to convert blunt ends to specific sticky ends.
with the complementary ends of other DNA fragments, Restriction enzymes can be used for mapping a DNA
directing the efficient joining of the cut ends. Because of fragment, as will be described in later sections. The col-
their ability to form hydrogen bonds with complemen- lection of fragments generated by digestion of a given
tary overhangs, these cuts are said to produce “sticky DNA fragment (e.g., a region of a human chromosome)
ends” at the cut site. Another mode of cutting separates with several restriction enzymes will be unique to that
the DNA duplex at the same place on both strands, DNA. This is the basis for forensic identification and
leaving flush, or blunt, ends. These ends can be rejoined paternity testing using restriction-fragment analysis of
as well, although not as efficiently as sticky ends. human DNA.
manipulate DNA in vitro,28 for instance, to make step-

Advanced Concepts wise deletions in linearized DNA or to modify DNA
ends after cutting with restriction enzymes. Exonu-
About half of all bacteria (and 90% of archeaea) cleases have different substrate requirements and will
have adaptive immune systems that function with therefore degrade specific types of DNA ends.
the restriction-modification systems. These clus- Exonuclease I from E. coli degrades single-stranded
tered, regularly interspaced, short palindromic DNA from the 3′-hydroxyl end into mononucleotides. Its
repeats (CRISPR)-Cas protein-based immune activity is optimal on long single-stranded ends, slowing
systems not only recognize exogenous DNA significantly as it approaches a double-stranded region.
but also maintain sequence records of previous It can be used to remove single-stranded excess primers
infections, similar to the function of vertebrate from double-stranded reaction products of DNA copying
immunity.25 or amplification procedures.
CRISPR is mediated by DNA sequence arrays Exonuclease III from E. coli removes 5′ mononucle-
flanked by Cas genes, the latter encoding nucleotides from the 3′ end of double-stranded DNA in the
ases and helicases. There are six types of CRISPR- presence of Mg2+ and Mn2+. It also has some endonu-
Cas structures, classified as class 1 (types I, III, clease activity, cutting DNA at apurinic sites. Exo III
and IV), which have multiple subunit complexes, removes nucleotides from blunt ends, recessed ends,
and class 2 (types II, V, and VI), which have and nicks but will not digest 3′ overhangs. Exo III was
single subunits.26 Foreign DNA is inserted into the used in the research setting to create nested deletions
host’s genome at these sequence arrays or spacers. in double-stranded DNA or to produce single-stranded
Sequences matching the host spacers are proto- DNA for dideoxy sequencing. Exo III can also promote
spacers. Protospacesrs are recognized by the host site-specific mutagenesis.
through a 2-5 nucleotide protospacer adjacent Exonuclease VII from E. coli digests single-stranded
motif (PAM) next to the protospacer sequence. DNA from either the 5′-phosphate or 3′-hydroxyl end. It
is one of the few enzymes with 5′ exonuclease activity.
Exo VII can be employed to remove long single strands
protruding from double-stranded DNA or single strands
DNA Ligase from mixtures of single- and double-stranded DNA.
DNA ligase catalyzes the formation of a phosphodiester
bond between adjacent 3′-hydroxyl and 5′-phosphoryl
nucleotide ends. Its existence was predicted by the
observation of replication, recombination, and repair
activities in vivo. These operations require reunion of
the DNA backbone after discontinuous replication on the
The initial analysis of the reaction joining sep-
lagging strand, strand exchange, or repair synthesis. In
arate DNA helices was performed with phys-
1967, DNA ligase was discovered in five different labo-
ically fractured DNA with no complementary
ratories.27 The isolated enzyme was found to catalyze the
bases at the ends of the fragments. The joining
end-to-end joining of separated strands of DNA.
reaction required the chance positioning of two
adjacent ends and was, therefore, not very effi-
Other DNA Metabolizing Enzymes cient. A better substrate for the enzyme would be
ends that could be held together before ligation
Nucleases
(i.e., by hydrogen bonds between single strands).
In contrast to endonucleases, exonucleases degrade H. Gobind Khorana showed that short synthetic
DNA from free 3′-hydroxyl or 5′-phosphate ends. Con- segments of DNA with single-strand complemen-
sequently, they will not work on closed circular DNA. tary overhangs joined into larger fragments effi-
These enzymes are used, under controlled conditions, to ciently.29 Several investigators noted the increased
efficiency of joining of ends of DNA molecules loops in duplex DNA. It was used in early nuclease
from certain bacterial viruses. These ends have mapping assays of DNA- and RNA-binding proteins.30
naturally occurring single-stranded overhangs. It recBC nuclease (Exonuclease V) from E. coli is an
was also observed that treatment of DNA ends ATP-dependent single- and double-stranded DNA nucle-
with terminal transferase to add short runs of A’s ase. Although it has no activity at nicks (short single-
to one fragment and T’s to another increased the strand gaps) in the DNA, it digests single-stranded DNA
efficiency of joining the ends of any two treated from either the 3′-hydroxyl or the 5′-phosphate end. It
fragments. Although not yet available when has some endonuclease activity on duplex DNA, gen-
ligase activity was being studied, the single- erating short fragments, or oligonucleotides. At high
strand overhangs left by some restriction enzymes levels of ATP, this enzyme also unwinds the DNA
were better substrates for DNA ligase than the double helix.
blunt ends due to hydrogen bonding of the com- Micrococcal nuclease digests single- and double-
plementary single-stranded bases. stranded DNA and RNA at AT- or AU-rich regions.
Although this enzyme can digest duplex DNA, it prefers
5′ … T C G A C T G C T A T … 3′ single-stranded substrates. It is used in the laboratory to
DNA
3′ …A G C T G A C G A T A … 5′ remove nucleic acid from crude extracts and also for the
analysis of chromatin structure.31
5′ … T C A T G C C C A C T A T G… 3′
Deoxyribonuclease I (DNAse I) from a bovine
3′ …A G T A C G G C C G A T A C… 5′
pancreas digests single- and double-stranded DNA at
5′ …G C A A T C A A A A A G T G C C… 3′
pyrimidines to oligonucleotides; so, technically, it is an
3′ …C G T T A G T T T T T C A C G G… 5′ endonuclease. It is used in both research and clinical
laboratories to remove DNA from RNA preparations.
Substrates for DNA ligase are broken double helices. Blunt DNAse I has also been used to detect exposed regions
ends (top) or noncomplementary overhangs (center) are joined
less efficiently than complementary overhangs (bottom). Also
of DNA in DNA protein-binding experiments.
note the complementary overhangs in Figure 1.13. DNA pol I from E. coli has exonuclease activity. For-
merly called exonuclease II, this activity is responsible
for the proofreading function of the polymerase.
Because nucleases are natural components of cellular
Nuclease Bal31 from Alteromonas espejiani can lysates, it is important to eliminate or inactivate them
degrade single- and double-stranded DNA from both when preparing nucleic acid specimens for clinical anal-
ends. It can also act as an endonuclease on single- ysis. Most DNA isolation procedures are designed to
stranded DNA. Because its activity at 20°C is slow minimize both endonuclease and exonuclease activity
enough to control with good resolution, it was useful in during DNA isolation. Purified DNA is often stored in
research applications to make nested deletions in DNA. TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) to
Mung bean nuclease from Mung bean sprouts digests chelate cations required by nucleases for activity.
single-stranded DNA and RNA. Because it leaves
double-stranded regions intact, it was used to remove
Helicases
overhangs from restriction fragments to produce blunt
ends for cloning. Its activity on RNA was used for DNA in bacteria and eukaryotes does not exist as the
transcriptional mapping and to resolve hairpins (folds) relaxed double helix as shown in Figure 1.1 but as a
in RNA. series of highly organized loops and coils. The release
S1 nuclease from Aspergillus oryzae and certain Neu- of DNA for transcription, replication, and recombination
rospora species is another single-strand–specific nucle- without tangling is brought about through cutting and
ase. It hydrolyzes single-stranded DNA or RNA into 5′ re-closing of the DNA sugar-phosphate backbone. These
mononucleotides. It also has endonuclease capability functions are carried out by a series of enzymes called
to hydrolyze single-stranded regions such as gaps and helicases.
As described with restriction endonucleases, the DNA double helix and not the other), as a means to differenti-
double helix can be broken apart by the separation of the ate host DNA from non-host and to provide resistance to
sugar-phosphate backbones in both strands, a double- restriction enzymes. Unlike prokaryotic DNA, eukary-
strand break. When only one backbone is broken (a otic DNA is methylated in specific regions. In eukary-
single-strand break or nick), the broken ends are free otes, DNA-binding proteins may limit accessibility or
to rotate around the intact strand. These ends can be guide methyltransferases to specific regions of the DNA.
digested by exonuclease activity or extended using the
intact strand as a template (nick translation). The nicking
and re-closing of DNA by helicases relieve topological
stress in highly compacted, or supercoiled, DNA as Histooricaal Higghlligghtts
required, for example, in advance of DNA replication
or transcription. Helicases are of two types: topoisom- Enzymatic interconversion of DNA forms was
erases and gyrases. Topoisomerases interconvert topo- first studied in vitro by observing the action
logical isomers or relax supertwisted DNA. Gyrases of two E. coli enzymes, topoisomerase I33 and
(type II topoisomerases) untangle DNA through dou- gyrase,34 on circular plasmids.
ble-strand breaks. They also separate linked rings of
DNA (concatemers).
Topoisomerases in eukaryotes have activity similar to
RECOMBINATION IN SEXUALLY
that in bacteria but with different mechanisms of cutting
and binding to the released ends of the DNA. Because of
REPRODUCING ORGANISMS
their importance in cell replication, topoisomerases are
Recombination is the mixture and assembly of new
the targets for several anticancer drugs, such as camp-
genetic combinations. Recombination occurs through
tothecin, the epipodophyllotoxins VP-16 and VM-26,
the molecular process of crossing over or physical
amsacrine, and the intercalating anthracycline derivatives
exchange between molecules. A recombinant mole-
doxorubicin and mitoxantrone.32 These topoisomerase
cule or organism is one that holds a new combination of
inhibitors bring about cell death by interfering with the
DNA sequences.
breaking and joining activities of the enzymes, in some
Based on Mendel’s laws, each generation of sexu-
cases trapping unfinished and broken intermediates.
ally reproducing organisms is a new combination of
the parental genomes. The mixing of genes generates
Methyltransferases
genetic diversity, increasing the opportunity for more
DNA methyltransferases catalyze the addition of methyl robust and well-adapted offspring. The beneficial effect
groups to nitrogen bases, usually adenines and cytosines of natural recombination is observed in the heterosis, or
in DNA strands. Most prokaryotic DNA is methylated, hybrid vigor, observed in genetically mixed or hybrid
or hemimethylated (methylated on one strand of the individuals compared with purebred organisms.
Histoorical Highlighhts
Early studies of recombination were done with progeny. These observations had been made before,
whole organisms. Mendel’s analysis of peas (Pisum with quantitative predictions of the probability of
species)35 established the general rules of recombi- phenotypes. Mendel proposed that traits are inherited
nation in sexually reproducing organisms. Mendel in a particulate manner, rather than blending as was
could infer the molecular exchange events that previously thought.
occurred in the plants by observing the phenotype of
What Mendel saw: What Mendel inferred:

phenotypes genotypes
RRGG ⫻ rrgg
Round Wrinkled
gray white
rg rg rg rg
Gamete production
and fertilization RG RGrg RGrg RGrg RGrg
RG RGrg RGrg RGrg RGrg

All round and gray
Self-pollinization
and fertilization
All RrGg
315 108 101 32

RrGg ⫻ RrGg
Round Round Wrinkled Wrinkled
gray white gray white
RG Rg rG rg
RG RRGG RRGg RrGG RrGg
Rg RRGg RRgg RrGg Rrgg
rG RrGG RrGg rrGG rrGg
rg RrGg Rrgg rrGg rrgg
Genotypic ratio:
1:1:2:2:4:2:2:1:1
Mendelian genetics showed that traits are inherited as unit characteristics. The probability of inheriting a given trait can then be calculated
from the traits of the parents. R, round; r, wrinkled; G, gray; g, white.
Sexually reproducing organisms mix genes in three

ways. First, at the beginning of meiosis, duplicated
chromosomes line up and recombine by crossing over
or breakage and reunion of the four DNA duplexes
(Fig. 1.14). This generates newly recombined duplexes
with genes from each duplicate. Then, the recombined
duplexes are randomly assorted into gametes (Fig. 1.15) Genetic recombination by crossing over Recombinant
so that each gamete contains one set of each of the chromosomes
recombined parental chromosomes. Finally, the gamete FIGURE 1.14 Generation of genetic diversity by crossing
will merge with a gamete from the other parent carrying over of homologous chromosomes.
Homologous
chromosomes
Recombined
chromosomes
Gametes
FIGURE 1.15 Recombined chromosomes are randomly assorted into gametes. Twenty-two other chromosomes will be randomly
assorted into the four gametes, giving each one a new collection of recombined chromosomes.
its own set of recombined chromosomes. The resulting in three ways: conjugation, transduction, and transfor-
offspring will contain a new set or recombination of the mation (Fig. 1.16).
genes of both parents. The nature of this recombination
is manifested in the combinations of inherited traits of
Conjugation
subsequent generations.
Recombinant DNA technology is a controlled mixing Bacteria that participate in conjugation are of two types,
of genes. Rather than relying on natural mixing of whole or sexes, termed F+ and F−. For conjugation to occur,
genomes, single genes can be altered, replaced, deleted, F− and F+ cells must be in contact with each other. The
or moved into new genomes. This directed diver- requirement for contact can be demonstrated by physi-
sity can produce organisms with predictable traits, as cally separating F+ and F− cells. If this is done, mating
natural purebreds, but with single-gene differences. The does not occur (Fig. 1.17). Microscopically, a filamen-
ability to manipulate single traits has implications not tous bridge is observed between mating bacteria. Work
only in the laboratory but also potentially in the treat- by J. Lederberg and William Hayes demonstrated polar-
ment and prevention of disease, for example, through ity in the conjugation process; that is, genetic informa-
gene therapy. tion could move from F+ to F− bacteria but not from
F− to F+ bacteria. The explanation for this was soon dis-
covered. The F+ bacteria had a “fertility factor” that not
RECOMBINATION IN ASEXUAL REPRODUCTION only carried the information from one cell to another but
also was responsible for establishing the physical con-
Movement and manipulation of genes in the labora- nection between the mating bacteria. The fertility factor
tory began with the study of natural recombination in was transferred from F+ to F− bacteria in the mating
asexually reproducing bacteria. Genetic information in process so that afterward, the F− bacteria became F+
asexually reproducing organisms can be recombined (Fig. 1.18).
Sexual reproduction Asexual reproduction
Donor cell Recipient cell

Gametes
Chromosome
Conjugation,
Zygote transduction, or
transformation
Duplicated
chromosomes,
recombination
Transformed
(recombinant) cell
FIGURE 1.16 Recombination in sexual (left) and asexual (right) reproduction.
F– Chromosome
Loss Conjugation
F+
F+
F–
Detachment Integration
Hfr F′
Filter impermeable
to bacteria Abnormal
detachment
FIGURE 1.17 Conjugating cells must be in physical contact
with each other (top) for successful transfer of the F+ pheno- FIGURE 1.18 Fertility (the ability to donate genetic informa-
type. If cells are separated by a membrane (bottom), tion) is controlled by the F factor. The F factor can exist by
F− bacteria do not become F+. itself or be integrated into the host chromosome.
Histooricaal Higghlligghtts F+. The F factor may be lost or cured during normal cell
division, converting an F+ bacteria to the F− state.
Historically, recombination was studied through The F factor can also insert itself into the host chro-
controlled mating and propagation of organisms. mosome through a crossover or recombination event.
George Beadle36 and others confirmed the con- Embedded in the chromosome, the F factor maintains
nection between the units of heredity and physical its ability to direct mating and can carry part or all of
phenotype using molds (Neurospora crassa), bac- the host chromosome with it across the mating bridge
teria, and viruses. Joshua Lederberg and Edward into F− bacteria. Strains with chromosomally embedded
L. Tatum37 demonstrated that bacteria mate and F factors are called high-frequency recombination (Hfr)
exchange genetic information to produce recom- bacteria. When the embedded F factor in these rarely
binant offspring. Lederberg and Tatum proved occurring strains pulls host chromosomal information
that genetic exchange between organisms was into recipient bacteria, another recombination event can
not restricted to the sexually reproducing molds. insert that information into the recipient chromosome,
These early studies first demonstrated the exis- forming a recombinant or new combination of genes of
tence of recombination in E. coli. the Hfr and F− bacteria. Hfr bacteria were used in the
first mapping studies.
E. coli
met + bio + Transduction

thr – leu –
In the early 1960s, Francois Jacob and Elie Wollman38
Requires threonine studied the transmission of units of heredity carried by
and leucine for growth thr + leu + viruses from one bacterium to another (transduction).
met + bio + Just as animal and plant viruses infect eukaryotic cells,
bacterial viruses, or bacteriophages, infect bacterial
met – bio – cells. The structure of bacteriophage T4 is one example
thr + leu + of the specialized protein coats that enable these viruses
to insert their DNA through the cell wall into the bacte-
Requires methionine
rial cell (Fig. 1.19). Alfred Hershey and Martha Chase
and biotin for growth
confirmed that the DNA of a bacterial virus was the
No growth on Growth on carrier of its genetic determination in the transduction
minimal medium minimal medium
process.39 Hershey and Chase used 35S to label the viral
(auxotrophic) (prototrophic)
protein and 32P to label the viral DNA. The experi-
Transfer of genetic information by conjugation can be demon-
ment showed that viral protein remained outside of the
strated using double mutants. Bacteria with double mutations
that require exogenous methionine and biotin (met and bio) or cell, whereas viral DNA entered the cell. Furthermore,
32
threonine and leucine (thr and leu) cannot grow on a medium P-labeled DNA could be detected in new viruses gen-
without addition of these nutrients (minimal medium). When erated in the transduction process (Fig. 1.20). Methods
these strains are mixed together, however, growth occurs. The soon developed using bacteriophages to move genetic
resulting bacteria have acquired the normal genes (+) through
information between bacteria by growing the phage on
transfer or conjugation.
one strain of bacteria and then infecting a second strain
with those viruses. Transduction is also useful in deter-
mining gene order. Seymour Benzer used transduction
The F factor was shown to be an extrachromosomal of the T4 bacterial virus to fine-map genes.40
circle of double-stranded DNA or plasmid carrying
the genes coding for construction of the mating bridge.
Genes carried on the F factor are transferred across the
Transformation
bridge and simultaneously replicated so that one copy of Although conjugation and transduction were the methods
the F factor remains in the F+ bacteria, and the other is for the initial study of the connection between DNA and
sent into the F− bacteria. After mating, both bacteria are phenotype, transformation, which was first observed in
1928 by Frederick Griffith,41 is the basis for modern-day and live rough-type bacteria, virulence returned. Further-
recombinant techniques. Griffith was investigating vir- more, he could recover live smooth-type bacteria from
ulence in Diplococcus (now known as Streptococcus) the dead mice. He concluded that something from the
pneumoniae. He had two strains of the bacteria: one dead smooth-type bacteria had “transformed” the rough-
with a rough colony type that was avirulent and one with type bacteria into the virulent smooth-type bacteria.
a smooth colony type that was virulent. Griffith intended What Griffith had observed was the transfer of DNA
to use these strains to develop a protective vaccine from one organism to another without the protection of
(Fig. 1.21). He knew that the live smooth-type bacteria a conjugative bridge or a viral coat. Fifteen years later,
were lethal in mice, and the live rough-type were not. If Oswald T. Avery, Colin MacLeod, and M. J. McCarty
he first killed the smooth-type bacteria by boiling them, identified the transforming material as DNA.42,43 They
virulence was lost, and they were no longer lethal to prepared boiled virulent bacterial cell lysates and sequen-
mice. Surprisingly, when he mixed killed smooth-type tially treated them with recently discovered enzymes
(Fig. 1.22). Protease and ribonuclease treatment,
which degraded protein and RNA, respectively, did not
Coat
Head
Rough type (avirulent)
DNA Mouse lives
Collar
Smooth type (virulent)
Mouse dies
Helical sheath
Heat-killed smooth type

Mouse lives
Base plate Tail fibers

Heat-killed smooth type
FIGURE 1.19 Bacteriophage T4 infects specific strains of E. plus rough type Mouse dies
coli. It has specialized structures. The tail fibers find the bacte-
rial surface and allow contact of the tail plate and injection of FIGURE 1.21 The “transforming factor” discovered by Grif-
the DNA in the viral head through the sheath into the fith was responsible for changing the phenotype of the aviru-
bacterium. lent rough-type bacteria to that of the virulent smooth type.
35S 32P
(protein coat) (DNA)
Host
cell
FIGURE 1.20 Radioactive protein does not enter the host cell during transduction (left). Radioactive DNA, however, does enter
and is passed to subsequent generations of viruses.
Circular plasmids
(several thousand Main circular chromosome
Cell lysate base pairs each) (4 million base pairs)
Transformation
+ proteinase Transformation
Antibiotic-resistance
genes
+ RNase Transformation Mobile

plasmid
Genes necessary for

DNA transfer
+ DNase Transformation
FIGURE 1.23 Plasmids are small extrachromosomal DNA
duplexes that can carry genetic information.
FIGURE 1.22 Avery, MacLeod, and McCarty showed that
destruction of protein or RNA in the cell lysate did not affect
the transforming factor. Only destruction of DNA prevented
transformation.
PLASMIDS
affect the transformation phenomenon that Griffith had DNA helices can assume both linear and circular forms.
demonstrated earlier. Treatment with deoxyribonuclease, Most bacterial chromosomes are in circular form, in
which degrades DNA, however, prevented transforma- contrast to chromosomes in higher organisms, such as
tion. They concluded that the “transforming factor” that fungi, plants, and animals, which are mostly linear. A
Griffith had first proposed was DNA. The transduction bacterial cell can contain, in addition to its own chro-
experiment of Alfred Hershey and Martha Chase also mosome complement, extrachromosomal entities, or
confirmed their findings that DNA carried genetic traits. plasmids (Fig. 1.23). Most plasmids are double-stranded
circles of 2,000 to 100,000 bp (2 to 100 kilobase pairs)
Advanced Concepts in size. Plasmids can carry genetic information. Due to
their size and effect on the host cell, plasmids carry only
Investigators performing early transformation a limited amount of information.
studies observed the transfer of broken chromo- The plasmid DNA duplex is compacted, or super-
somal DNA from one population of bacterial cells coiled. Breaking one strand of the plasmid duplex, or
to another. Naked DNA transferred in this way, nicking, will relax the supercoil (Fig. 1.24), whereas
however, is very inefficient. Unprotected DNA is breaking both strands will linearize the plasmid. Dif-
subject to physical shearing as well as chemical ferent physical states of the plasmid DNA can be
degradation from naturally occurring nucleases, resolved by distinct migration characteristics during gel
especially on the broken ends of the DNA mol- electrophoresis.
ecules. Natural transformations are much more Plasmids were found to be a source of resistant phe-
efficient because the transforming DNA is in the notypes in multidrug-resistant bacteria.44 The demon-
form of circles or otherwise protected, especially stration that multiple drug resistance in bacteria can be
on the ends. eliminated by treatment with acridine dyes45 was the
first indication of the episomal (plasmid) nature of the
Locally denatured daughter cells at cell division or uptake by host cells in

base pairs transformation.
Advanced Concepts
Gyrase + ATP The circular nature of R factors was demonstrated
by buoyant-density centrifugation.47 Plasmid DNA
Topoisomerase has a density higher than that of the host chromo-
some and can be isolated from separate, or sat-
ellite, bands in the gradient. Examination of the
fractions of the higher-density DNA reveals small
circular species. These circles are absent from
drug-sensitive bacteria.
Relaxed circle Supercoiled DNA
FIGURE 1.24 Supercoiled plasmids can be relaxed by Compared with fragments of DNA, plasmids are more
nicking (left) or by local unwinding of the double helix (right). efficient vehicles for the transfer of genes from one cell
to another. Upon cell lysis, supercoiled plasmids can
enter other cells more efficiently. Plasmids are used
resistance factor, similar to the F factor in conjugation.
in recombinant DNA technology to introduce specific
(Acridine dyes induce the loss of episomes.) In resistant
traits. By manipulation of the plasmid DNA in vitro,
bacteria, plasmids carrying the genes for inactivation or
specific genes can be introduced into cells to produce
circumvention of antibiotic action were called resistance
new phenotypes or recombinant organisms. The ability
transfer factors (RTF), or R factors. R factors promote
to express genetic traits from plasmids makes it possi-
resistance to common antibiotics such as chloramphen-
ble to manipulate phenotype in specific ways. As will
icol, tetracycline, ampicillin, and streptomycin. Another
be described in later chapters, plasmids play a key role
class of plasmid, colicinogenic factors, carries resistance
in the development of the procedures used in molecular
to bacteriocins, toxic proteins manufactured by bacteria.
analysis.
The acquisition of the resistance genes from host chro-
mosomes of unknown bacteria is the presumed origin
of these resistance factors.46 Drug-resistance genes are
commonly gained and lost from episomes in a bacte- Advanced Concepts
rial population. Two different R factors in a single cell
can undergo a recombination event, producing a new, Plasmids are found not only in bacteria but in multi-
recombinant plasmid with a new combination of resis- cellular plants and animals as well. Some viruses,
tance genes. such as the single-stranded DNA virus M13, have
Plasmids were initially classified into two general a transient plasmid phase in their life cycle. Lab-
types: large plasmids and small plasmids. Large plas- oratory techniques requiring single-stranded ver-
mids include the F factor and some of the R plasmids. sions of specific DNA sequences were once based
Large plasmids carry genes for their own transfer and on the manipulation of the plasmid (duplex circle)
propagation and are self-transmissible. Large plasmids phase of these viruses and isolation of recombi-
occur in small numbers, one or two copies per chromo- nant single-stranded circles from the virus. This
some equivalent. Small plasmids are more numerous in technology was used in methods devised to deter-
the cell, about 20 copies per chromosomal equivalent; mine the order or sequence of nucleotides in the
however, they do not carry genes directing their main- DNA chain.
tenance. They rely on high numbers for distribution into
O O
C C
H3C C NH HC NH
OR OR
O– P O HC C O O– P O HC C O
N N
O O O O
H2C CH CH H2C CH CH
HC CH2 HC CH
OH OH OH
dT U
FIGURE 1.25 Uracil (U), the nucleotide base that replaces thymine in RNA, has the pyrimidine ring structure of thymine (dT)
minus the methyl group. Uracil forms hydrogen bonds with adenine.
RNA
viruses, the retroviruses, which include leukemia
Ribonucleic acid (RNA) is a polymer of nucleotides viruses and the human immunodeficiency virus,
similar to DNA. It differs from DNA in the sugar moi- have RNA genomes, and in order to replicate using
eties, having ribose instead of deoxyribose and, in host-cell machinery, they must first make a DNA
one nitrogen base component, having uracil instead of copy of their genome by reverse transcription.
thymine (thymine is 5-methyl uracil; Fig. 1.25). Further-
more, RNA is synthesized as a single strand rather than
as a double helix. Although almost all RNA strands do
not have complementary partner strands, they are not
Transcription
completely single stranded. Through internal homol-
ogies, RNA species fold and loop upon themselves to DNA can only store information. In order for this infor-
take on a double-stranded character that is important mation to be utilized, it must be transcribed and then
for their function. RNA can also pair with complemen- translated into protein, a process called gene expres-
tary single strands of DNA or another RNA and form a sion. A specific type of RNA, messenger RNA (mRNA),
double helix. carries the information in DNA to the ribosomes, where
There are several types of RNAs found in the cell. it is translated into protein.
Ribosomal RNA, messenger RNA, transfer RNA, and Transcription is the copying of one strand of DNA
small nuclear RNAs have distinct cellular functions. into RNA by a process similar to that of DNA repli-
RNA is copied, or transcribed, from DNA. cation. This activity, catalyzed by RNA polymerase,
occurs mostly in interphase. Whereas a single type of
RNA polymerase catalyzes the synthesis of all RNA in
Advanced Concepts most prokaryotes, there are three types of RNA poly-
merases in eukaryotes: RNA polymerase pol I, pol II,
Evolutionary theory places RNA as the original and pol III. Pol I and III synthesize noncoding RNA
genetic material from which DNA has evolved. In (Table 1.3).48 Pol II is responsible for the synthesis of
most organisms, RNA is an intermediate between messenger RNA (mRNA), the type of RNA that carries
the storage system of DNA and the proteins genetic information to be translated into protein. Evi-
responsible for phenotype. One family of RNA dence suggests that transcription takes place at discrete
stations of the nucleus into which the DNA molecules
Transcription Initiation
TABLE 1.3 RNA Polymerases
RNA polymerase and its supporting accessory proteins
Enzyme Template Product assemble on DNA at a specific site called the promoter.
Initiation sites of transcription (RNA synthesis) greatly
E. coli RNA polymerase II DNA mRNA outnumber DNA initiation sites in both prokaryotes and
RNA polymerase I DNA rRNA eukaryotes. There are also many more molecules of RNA
polymerase than DNA polymerase in the cell. Although
RNA polymerase II DNA mRNA functionally catalyzing the same reaction, RNA poly-
RNA polymerase III DNA tRNA, snRNA merases in prokaryotes and eukaryotes differ and work
with different supporting proteins to find and bind to
Mitochondrial RNA DNA mRNA DNA in preparation for transcription. In prokaryotes,
polymerase a basal transcription complex comprised of the large
Mammalian DNA DNA Primers and small subunits of RNA polymerase and additional
polymerase α sigma factors assembles at the start site. The eukaryotic
transcription complex requires RNA polymerase and up
HCV RNA polymerase RNA Viral genome
to 20 additional factors for accurate initiation. Initiation
Dengue virus RNA RNA Viral genome of RNA synthesis is regulated in all organisms so that
polymerase genes are transcribed as required by specific cell types.
PolyA polymerase None PolyA tails
The regulation of transcription differs in prokaryotes and
eukaryotes.
TABLE 1.4 Cellular Location and Activity Transcription Elongation

of RNA Pol I, II, and III in Eukaryotes RNA polymerases in both eukaryotes and prokaryotes
synthesize RNA using the base sequence of one strand
Type Location Products α-Amanitin of the double helix as a guide (Fig. 1.26). The comple-
mentary strand of the DNA template (that is not copied)
I Nucleolus 18s, 5.8s, 28s Insensitive
rRNA has a sequence identical to that of the RNA product
(except for the U for T substitution in RNA).
II Nucleus mRNA, snRNA Inhibited RNA polymerases work more slowly than DNA
III Nucleus tRNA, 5s rRNA Inhibited by high
polymerases (50 to 100 bases/sec for RNA synthesis
concentration vs. 1,000 bases/sec for DNA replication) and with less
fidelity. The DNA double helix is locally unwound into
move (Table 1.4).2 One of these sites, the nucleolus, is

the location of ribosomal RNA synthesis. RNA
mRNA polymerase
Advanced Concepts
DNA 3′ 5′
5′ 3′
DNA must be released locally from histones and
the helix unwound in order for transcription to
occur. These processes involve the participation Direction of transcription
of numerous factors, including DNA-binding pro- FIGURE 1.26 RNA polymerase uses one strand of the double
teins, transcription factors, histone-modification helix (the antisense strand) as a template for synthesis of RNA.
enzymes, and RNA polymerase. About 10 base pairs of DNA are unwound or opened to allow
the polymerase to work.
single strands to allow the assembly and passage of the TYPES/STRUCTURES OF RNA
transcription machinery, forming a transcription bubble.
Unlike DNA synthesis, RNA synthesis does not There are several types of RNAs found in the cell. Ribo-
require priming. The first ribonucleoside triphosphate somal RNA, mRNA, transfer RNA, and small nuclear
retains all of its phosphate groups as the RNA is poly- RNAs have distinct cellular functions.
merized in the 5′ to 3′ direction. Subsequent ribonucleo-
side triphosphates retain only the alpha phosphate, the
one closest to the ribose sugar. The other two phosphate Ribosomal RNA
groups are released as orthophosphate during the syn-
The largest component of cellular RNA is ribosomal
thesis reaction.
RNA (rRNA), comprising 80% to 90% of the total cel-
lular RNA. The various types of ribosomal RNAs are
Transcription Termination named for their sedimentation coefficient (S) in density-
gradient centrifugation.52 rRNA is an important structural
RNA synthesis terminates differently in prokaryotes
and functional part of the ribosomes, cellular organelles
and eukaryotes. In prokaryotes, some RNA synthesis
where proteins are synthesized (Fig. 1.27).
is responsive to protein products, such that high levels
In prokaryotes, there are three rRNA species, the 16S
of a gene product induce termination of its own syn-
found in the ribosome small subunit and the 23S and
thesis. Termination is accomplished in some genes by
5S found in the ribosome large subunit, all synthesized
interactions between RNA polymerase and nucleotide
from the same gene. In eukaryotes, rRNA is synthesized
signals in the DNA template. In other genes, an addi-
from highly repeated gene clusters. Eukaryotic rRNA is
tional factor, rho, is required for termination. Rho is a
copied from DNA as a single 45S precursor RNA (pre-
helicase enzyme that associates with RNA polymerase
ribosomal RNA) that is subsequently processed into the
and inactivates the elongation complex at a cytosine-rich
18S species of the ribosome small subunit and 5.8S and
termination site in the DNA.49,50 Rho-independent termi-
28S species of the large subunit. Another rRNA species,
nation occurs at G:C-rich regions in the DNA, followed
5S, found in the large ribosome subunit in eukaryotes, is
by A:T-rich regions. The G:C bases are transcribed into
synthesized separately.
RNA and fold into a short double-stranded hairpin,
which slows the elongation complex. The elongation
complex then dissociates as it reaches the A:T-rich area.
Messenger RNA
In eukaryotes, mRNA synthesis, catalyzed by pol II,
proceeds along the DNA template until a polyadenyla- Messenger RNA (mRNA) is the initial connection
tion signal (polyA site) is encountered. At this point, between the information stored in DNA and the trans-
the process of termination of transcription is activated. lation apparatus that will ultimately produce the protein
There is no specific sequence in DNA that specifies ter- products responsible for the phenotype. In prokaryotes,
mination of transcription. As the polymerase proceeds mRNA is synthesized and simultaneously translated
past the polyA site, the nascent mRNA is released by into protein. Prokaryotic mRNA is sometimes poly-
an endonuclease associated with the carboxy terminal cistronic; that is, one mRNA codes for more than one
end of pol II. RNA synthesized beyond the site trails out protein. Eukaryotic mRNA, in contrast, is monocis-
of the polymerase and is bound by another exonuclease tronic, having only one protein per mRNA. Eukaryotes
that begins to degrade the RNA 5′ to 3′ toward the RNA can, however, produce different proteins from the same
polymerase. When the exonuclease catches up with the DNA sequences by starting the RNA synthesis in dif-
polymerase, transcription stops. ferent places or by processing the mRNA differently. In
Pol I in eukaryotes terminates transcription just prior eukaryotes, copying of RNA from DNA and protein syn-
to a site in the DNA (Sal box) with the cooperation of a thesis from the RNA are separated by the nuclear mem-
termination factor, TTF1.51 The pol III termination signal brane barrier. Eukaryotic mRNA undergoes a series of
is a run of adenine residues in the template. Pol III tran- post-transcriptional processing events before it is trans-
scription termination requires a termination factor. lated into protein (Fig. 1.28).
Prokaryotic ribosome Eukaryotic ribosome
Large subunit Large subunit

23S rRNA 28S rRNA
5S rRNA 5S rRNA
50S 5.8S rRNA 60S
L1–L31
L1–L50
70S 80S
Small subunit
16S rRNA Small subunit
18S rRNA
30S
40S
S1–S21
S1–S32
FIGURE 1.27 Prokaryote and eukaryote ribosomal subunits are of similar structure but different size. Ribosomal RNAs (left) are
assembled with 52 or 82 ribosomal proteins (center) to make the subunits that will form the complete ribosome in association with
mRNA.
Promoter Exon 1 Intron 1 Exon 2 Intron 2 Exon 3
5′ 3′
3′ 5′
DNA
Transcription
Pre-mRNA 5′ 3′
Processing
mRNA 7 Me G AAAAA
5′ 3′
FIGURE 1.28 DNA (top) and heteronuclear RNA (middle) contain intervening (intron) and expressed (exon) sequences. The
introns are removed, and the mature RNA is capped and polyadenylated (bottom).
Advanced Concepts
The amount of a particular mRNA in a cell is related
to the requirement for its final product. Some messages The secondary structure of rRNA is important for
are transcribed constantly and are relatively abundant in the integrity and function of the ribosome. Not
the cell (constitutive transcription), whereas others are only is it important for ribosomal structure, but it
transcribed only at certain times during the cell cycle is also involved in the correct positioning of the
or under particular conditions (inducible, or regulatory, ribosome on the mRNA and with the transfer RNA
transcription). Even the most abundantly transcribed during protein synthesis.53,54
mRNAs are much less plentiful in the cell than rRNA.
Messenger RNA Processing and Hogness.59 In these experiments, mRNA and

Polyadenylation duplex genomic DNA were incubated together at
The study of mRNA in eukaryotes was facilitated by conditions under which the more stable RNA–
the discovery that most messengers carry a sequence DNA hybrids are favored over the DNA duplexes.
of polyadenylic acid at the 3′ terminus, a polyA tail. The resulting structures released loops of unpaired
The run of adenines was first discovered by hydrogen DNA (introns). The DNA duplexed with RNA
bonding of mRNA to polydeoxythymine on poly(dT) corresponded to the coding sequences (exons).
cellulose.55 Polyuridine or polythymine residues cova-
lently attached to cellulose or sepharose substrates are Capping
often used to specifically isolate mRNA in the laboratory. Eukaryotic mRNA is blocked at the 5′ terminus by
The polyA tail is not coded in genomic DNA. It is a 5′-5′ pyrophosphate bridge to a methylated guano-
added to the RNA after synthesis of the pre-mRNA. sine.60 The structure is called a cap. The cap is a 5′-5′
A protein complex recognizes the RNA sequence, pyrophosphate linkage of 7-methyl guanosine to either
AAUAAA, and cleaves the RNA chain 11 to 30 bases 3′ 2′ O-methyl guanine or 2′ O-methyl adenine of the
to that site. The enzyme that cuts pre-mRNA in advance mRNA:
of polyadenylation has not been identified. Recent 7 -methyl
G 5′ + pNpNp5′ + 2′O -methyl (G or A )pNpNpNp…
studies suggest that a component of the protein complex
related to the system that is responsible for removing where p represents a phosphate group, and N represents
the 3′ extension from pretransfer RNAs may also be any nucleotide.
involved in the generation of the 3′ ends on mRNA.56,57
The enzyme polyadenylate polymerase is responsible Advanced Concepts
for adding the adenines to the end of the transcript. A
run of up to 200 nucleotides of polyA is typically found About 30% of the mRNAs, notably histone
on mRNA in mammalian cells. mRNAs, are not polyadenylated.61 The functional
differences between polyA+ and polyA− mRNA
are not clear. The polyA tail may be involved in
Histooricaal Higghlligghtts the movement of the mRNA from the nucleus to
the cytoplasm, association with other cell com-
A DNA copy (complementary DNA, copy DNA
ponents, the maintenance of secondary structure,
[cDNA]) of mature mRNA can be made by reverse
or the stability of the molecule. Abnormalities in
transcription of mRNA (synthesis of DNA from
the 3′-end processing mechanism have been found
the mRNA template). Compared with the original
in oncological, immunological, neurological, and
gene on the chromosome, the cDNA version of
hematological diseases.62
eukaryotic genes is smaller than the chromosomal
version. Restriction enzyme mapping confirms
The cap confers a protective function and serves as a
that this is not due to premature termination of
recognition signal for the translational apparatus. Caps
the genomic transcript. The entire functional gene
differ with respect to the methylation of the end nucle-
is present in the shorter sequence because cDNA
otide of the mRNA. In some cases, 2′ O-methylation
versions of eukaryotic genes can be expressed
occurs not only on the first but also on the second nucle-
(transcribed and translated) into complete pro-
otide from the cap. Other caps methylate the first three
teins. The chromosomal version of the gene must,
nucleotides of the RNA molecule.
therefore, have extra sequences inserted between
the protein-coding sequences. The direct location Splicing
and size of these intervening sequences or introns Prokaryotic structural genes contain uninterrupted
were first demonstrated by electron microscopy lengths of open reading frame, sequences that code
of hybrids between mRNA and cDNA58 using the for amino acids. In contrast, eukaryotic coding regions
method of R-loop mapping developed by White are interrupted with long stretches of noncoding DNA
following consensus sequences for the donor and

Exon Intron Exon acceptor splice junctions of group I, group II, and
5′ AG GU A AGU UAUAC NCAG G 3′ nuclear introns:65
( intron )
branch site donor site acceptor site
U GAAUG GA 5′
A G // G U A A G U Y N C U R A C YN N C A G // G
UAUAC NCAG G 3′
The branch-point sequence YNCURAC is variable in
mammals but almost invariant in the yeast Saccharomy-
UG
A 5′ AG 3′
ces cerevisiae (UACUAAC).
A
U
G
UAUAC NCAG 3′ 5′ G 3′ Advanced Concepts
Discarded intron
Caps are present on all eukaryotic mRNA bound
5′ AG G 3′ for translation, with the exception of some mRNA
transcribed from mitochondrial DNA. Capping
FIGURE 1.29 RNA splicing at the 5′ splice site (AGGU-
occurs after initiation of transcription, catalyzed by
AAGU), branch (UAUAC), and 3′ splice site (NCAGG) con-
sensus sequences. The intron is removed through a
the enzyme guanylyltransferase. This enzyme links
transesterification reaction involving a guanine nucleotide of a guanosine monophosphate provided by guano-
the 5′ site and an adenine in the branch sequence. The product sine triphosphate to the 5′ phosphate terminus of
of this reaction is the discarded intron in a lariat structure. the RNA with the release of pyrophosphate. In
Another transesterification reaction connects the exons. some viruses, guanosine diphosphate provides the
guanidine residue, and monophosphate is released.
(intervening) sequences called introns. Newly tran- Caps of mRNA are recognized by ribosomes just
scribed mRNA, heteronuclear RNA (hnRNA), is much before translation.66
larger than mature mRNA because it still contains the
intervening sequences. Labeling studies demonstrated
that the hnRNA is capped and tailed and that these mod- There are four types of introns—group I, group II,
ifications survive the transition from hnRNA to mRNA, nuclear, and tRNA—depending on the mechanism of
which is simply a process of removing the interven- their removal from hnRNA. Group I introns are found in
ing sequences from the hnRNA. Introns are removed nuclear, mitochondrial, and chloroplast genes. Group II
from hnRNA by splicing (Fig. 1.29). The remaining introns are found in mitochondrial and chloroplast genes.
sequences that code for the protein product are exons. Group I introns require a guanosine-triphosphate mole-
cule to make a nucleophilic attack on the 5′ phosphate
Histooricaal Higghlligghtts of the 5′ end of the intron. This leaves a 3′ OH at the
end of the 5′ exon (splice donor site), which attacks the
Although removal of all nuclear introns requires 5′ end of the next exon (splice acceptor site), forming
protein catalysts, some introns are removed a new phosphodiester bond and releasing the interven-
without the participation of protein factors in ing sequence. Group II introns are removed in a similar
a self-splicing reaction. The discovery of self- reaction initiated by the 2′ OH of an adenosine within
splicing was the first demonstration that RNA the intron attacking the 5′ phosphate at the splice donor
could act as an enzyme.63,64 site. When the 3′ OH of the splice donor site bonds with
Inspection of splice junctions from several the splice acceptor site of the next exon, the intervening
organisms and genes has demonstrated the sequence is released as a lariat structure (see Fig. 1.29).
This lariat contains an unusual 2′, 3′, and 5′ triply linked

nucleotide, the presence of which proved the mecha- TABLE 1.5 Small Nuclear RNA Isolated
nism. Removal of nuclear introns occurs by the same From HeLa Cervical Carcinoma and Novikoff
transesterification mechanism, except this reaction is Hepatoma Cells17
catalyzed by specialized RNA–protein complexes (small
Species Approximate
nucleoprotein particles). These complexes contain the
Species (HeLa) (Novikoff ) Length (Bases)
small nuclear RNAs U1, U2, U4, U5, and U6.
Why are eukaryotic genes interrupted by introns? SnA U5 180
Splicing may be important for the timing of the transla-
SnB 210
tion of mRNA in the cytoplasm. One theory holds that
introns evolved as a means of increasing recombina- SnC U2 196
tion frequency within genes as well as between genes
without breaking coding sequences. The discontinuous SnD U1B 171
nature of eukaryotic genes may also protect the coding SnE/ScE (5.8S rRNA) 5.8S
regions from genetic damage by toxins or radiation.
Alternative splicing modifies products of genes by SnF U1A 125
alternate insertion of different exons. For example, the SnG/ScG (5S rRNA) 5S I and II
production of calcitonin in the thyroid or calcitonin
gene-related peptide in the brain depends on the exons SnG′ 5S III 120
included in the mature mRNA in these tissues.67 Alterna- SnH 4.5S I, II, and III 96
tive splicing has been found in over 40 different genes.
SnI (tRNA)
SnK 260
Advanced Concepts
SnP 130
The splicing of transfer RNA (tRNA) transcripts
ScL (viral 7S) 260
involves breakage and reunion of the RNA chain.
Endonucleases cleave the tRNA precisely at ScM 180
the intron ends. The resulting tRNA ends, a 2′,
ScD 180
3′ cyclic phosphate and a 5′ OH, are then ligated
in a complex reaction that requires ATP, followed
by further base modification in some tRNAs.
after its transcription by RNA polymerase I or III. These
RNAs sediment in a range of 6 to 8S. Small nuclear
Abnormalities in the splicing process are responsible RNAs isolated from hepatoma and cervical carcinoma
for several disease states. Some β-thalassemias result cell lines are summarized in Table 1.5.
from mutations in the splice recognition sequences of
the β-globin genes. Certain autoimmune conditions
Transfer RNA
result from production of antibodies to RNA–protein
complexes. Autoantibodies against U1 RNA, one of the Translation of information from nucleic acid to protein
small nuclear RNAs required for splicing, are associated requires reading of the mRNA by ribosomes, using
with systemic lupus erythematosus. adaptor molecules or transfer RNA (tRNA). Transfer
RNAs are relatively short, single-stranded polynucle-
otides of 73 to 93 bases in length, MW 24,000 to 31,000.
Small Nuclear RNA
There is at least one tRNA for each amino acid.
Small nuclear RNA (snRNA) functions in splicing in Eight or more of the nucleotide bases in all tRNAs are
eukaryotes. Small nuclear RNA stays in the nucleus modified, usually methylated, after the tRNA synthesis.
3′ Amino acid
A O H
C CCA terminus
C C N2H
C Acceptor end
O R
5′ A 3′
G C 5′
G C
G U Acceptor arm Acceptor arm
C G
G C
D loop U U TyCC loop TyCC loop
G C U
U A G G C C U A
D G A mG G
C U G C G C
U C C G G T
G C D stem
G D A G C G C mG D
2
C G A G D loop
G
U A
C G Anticodon stem Variable loop
Anticodon loop C G
C G
U
U m1
Anticodon loop
G C
Anticodon Anticodon
FIGURE 1.30 Alanine tRNA is an example of the general structure of tRNA, which is often depicted in a cruciform structure
(left). The inverted “L” (right) more accurately depicts the structure formed by intrastrand hydrogen bonding.
Most tRNAs have a guanylic residue at the 5′ end and the Advanced Concepts
sequence CCA at the 3′ end. Through intrastrand hybrid-
ization, tRNAs take on a cruciform structure of four Small nuclear RNAs serve mostly a structural role
to five double-stranded stems and three to four single- in the processing of mRNA. Several of a family of
stranded loops (Fig. 1.30). The CCA at the 3′ end of proteins (Sm proteins) assemble into a (60 Å by
the tRNA is where the amino acid will be covalently 30 to 40 Å) doughnut-shaped complex that inter-
attached to the tRNA. A seven-base loop (the TΨC loop, acts with the U-rich regions of poly (U) RNAs.68,69
where Ψ stands for the modified nucleotide pseudouri- U1 RNA is complementary to sequences at the
dine) contains the sequence 5′-TΨCG-3′. The variable splice donor site, and its binding distinguishes
loop is larger in longer tRNAs. Another seven-base loop the sequence GU in the splice site from other GU
(the anticodon loop) contains the three-base-pair anti- sequences in the RNA. U2 RNA recognizes the
codon that is complementary to the mRNA codon of splice acceptor site. In lower eukaryotes, another
its cognate amino acid. An 8- to 12-base loop (D loop) protein binds to the branch-point sequence, initi-
is relatively rich in dihydrouridine, another modified ating further protein assembly and association of
nucleotide. Studies of pure crystalline tRNA using x-ray U4, U5, and U6, with the looped RNA forming a
diffraction reveal that the cruciform secondary struc- complex, the spliceosome, in which the transester-
ture of tRNA takes on an additional level of hydrogen ification reaction linking the exons together takes
bonding between the D loop and the TΨC loop to form place.
a γ-shaped structure (see Fig. 1.30).
RNA POLYMERASES
Advanced Concepts
RNA synthesis is catalyzed by RNA polymerase
Mitochondria contain distinct, somewhat smaller, enzymes (Table 1.4). One multisubunit prokaryotic
tRNAs. enzyme is responsible for the synthesis of all types of
RNA in the prokaryotic cell. Eukaryotes have three dif-
ferent RNA polymerase enzymes. All of these enzymes
are DNA-dependent RNA polymerases; that is, they
require a DNA template. In contrast, RNA-dependent
Advanced Concepts RNA polymerases require an RNA template.
Bacterial RNA polymerase consists of five subunits,
The tRNA genes contain 14 to 20 extra nucleotides two α and one of each β, β′, and σ (Fig. 1.31).71
in the sequences coding for the anticodon loop that
are transcribed into the tRNA. Enzymes that rec-
ognize other tRNA modifications remove these
sequences (introns) by a cleavage-ligation process. Histooricaal Higghlligghtts
Intron removal, the addition of CCA to the 3′ end,
and nucleotide modifications all occur following The α2, β, β′ core enzyme retains the catalytic
tRNA transcription. Enzymatic activities responsi- activity of the α2, β′, σ complete enzyme, or holo-
ble for intron removal and the addition of CCA enzyme, suggesting that the sigma factor plays
may also contribute to intron removal and polyA no role in RNA elongation.72 In fact, the sigma
addition to mRNA. factor is released at RNA initiation. The role of
the sigma factor is to guide the complete enzyme
to the proper site of initiation on the DNA.
In 1964, Robert Holley and colleagues at Cornell
University solved the first tRNA sequence. The β′
sequence was that of alanine tRNA of yeast.70 α
Yeast tRNAala is 76 bases long; 10 of these bases α β
Core enzyme (α,α2,β,β′)
are modified.
β′
α
α β
Other RNAs Holoenzyme (α,α2,β,β′,σ)
σ
Since the late 1990s, increasing varieties of RNA species
have been described in prokaryotes and eukaryotes. In
addition to RNA synthesis and processing, these mol- ρ Rho termination factor (ρ)
ecules influence numerous cellular processes, including
plasmid replication, bacteriophage development, chro- FIGURE 1.31 Prokaryotic RNA polymerase is made up of
mosome structure, and development. Untranslated RNA separate proteins. The four subunits that make up the core
molecules have been termed sRNAs in bacteria and enzyme have the capacity to synthesize RNA. The sigma
noncoding RNAs (ncRNAs) in eukaryotes. Their role in cofactor aids in the accurate initiation of RNA synthesis. The
controlling phenotype will be discussed in Chapter 2. rho cofactor aids in termination of RNA synthesis.
Advanced Concepts Advanced Concepts

The nucleotide sequence of mRNA can be altered The most well-studied eukaryotic RNA poly-
after RNA synthesis by RNA editing. There are merase is pol II from the yeast S. cerevisiae. It
two mechanisms of RNA editing in humans. In is a 0.4-megadalton complex of 12 subunits. The
one, such enzymes as cytidine deaminase and ade- yeast enzyme works in conjunction with a large
nosine deaminase convert C to U and A to inosine complex of proteins required for promoter recog-
(read as G), respectively. These enzymes recognition and melting, transcription initiation, elon-
nize specific sequences in the mRNA. The other gation and termination, and transcript processing
mechanism involves guide RNA (gRNA), a small (splicing, capping, and polyadenylation).
nuclear RNA that hydrogen bonds to the mRNA
transcript and mediates the addition or removal of
bases from the mRNA. The former substitution
mechanism is responsible for RNA editing of the RNA viruses carry their own RNA-dependent RNA
APOB gene so that different versions are made in polymerases. Hepatitis C virus, Zika virus, and Dengue
the liver and intestines. virus carry this type of polymerase to replicate their RNA
genomes. RNA-dependent RNA polymerase activity has
also been found in plants and lower eukaryotes, where
the purpose of these enzymes in cells may be associated
In eukaryotes, there are three multisubunit RNA poly- with gene silencing.76 The enzyme activity may serve as
merases, RNA polymerase I, II, and III (Table 1.5), a therapeutic target for antiviral agents.77
and a single-subunit mitochondrial RNA polymerase PolyA polymerase is a template-independent RNA
imported to organelles. The three RNA polymerases in polymerase. This enzyme adds adenine nucleotides to the
eukaryotic cells were first distinguished by their loca- 3′ end of mRNA.78 The resulting polyA tail is important
tions in the cell. RNA polymerase I (pol I) is found in for mRNA stability and translation into protein.
the nucleolus. RNA polymerase II (pol II) is found in the
nucleus. RNA polymerase III (pol III), one of the first
nucleic acid polymerases discovered, is also found in the OTHER RNA-METABOLIZING ENZYMES
nucleus and sometimes in the cytoplasm.73
Ribonucleases
Ribonucleases degrade RNA in a manner similar to the
degradation of DNA by deoxyribonucleases (Table 1.6).
Advanced Concepts There are several types of ribonucleases, generally clas-
sified as endoribonucleases and exoribonucleases.
The three polymerases were also distinguished Endonucleases include RNase H, which digests the
by their differential sensitivity to the toxin α- RNA strand in a DNA–RNA hybrid; RNase I, which
amanitin.74,75 This toxin is isolated from the poi- cleaves single-stranded RNA; and RNase III, which
sonous mushroom Amanita phalloides. Pol II is digests double-stranded RNA. RNase P removes precur-
sensitive to relatively low amounts of this toxin, sor nucleotides from tRNA molecules. RNase A, RNase
pol III is sensitive to intermediate levels, and pol T1, and RNase T2 cleave single-stranded RNA at spe-
I is resistant. α-Amanitin was invaluable in the cific residues. A combination of RNase A, T1, and T2 is
research setting to determine which polymerase used in some laboratory procedures investigating gene
activity is responsible for the synthesis of newly expression and transcript structure. An endoribonuclease
discovered types of RNA and to dissect the bio- formed by cleavage and polyadenylation-specific factor
chemical properties of the polymerases. (CPSF) and other factors that bind to the polyA site are
required for proper termination of RNA synthesis in
TABLE 1.6 RNases Used in Laboratory Procedures82
Enzyme Source Type Substrate
RNase A Bovine Endoribonuclease Single-stranded RNA 3′+ to pyrimidine residues
RNase T1 Aspergillus Endoribonuclease 3′ Phosphate groups of guanines
RNase H E. coli Exoribonuclease RNA hybridized to DNA
RNase CL3 Gallus Endoribonuclease RNA next to cytidylic acid
Cereus RNase Physarum Endoribonuclease Cytosine and uracil residues in RNA
RNase Phy M Physarum Endoribonuclease Uracil, adenine, and guanine residues in RNA
RNase U2 Ustilago Endoribonuclease 3′ Phosphodiester bonds next to purines
RNase T2 Aspergillus Endoribonuclease All phosphodiester bonds, preferably next to adenines
S1 nuclease Aspergillus Exoribonuclease RNA or single-stranded DNA
Mung bean nuclease Mung bean sprouts Exoribonuclease RNA or single-stranded DNA
RNase Phy I Physarum Exoribonuclease Guanine, adenine, or uracil residues in RNA
mammals.79 Along with RNA polymerase II subunits RNA. These enzymes have been characterized in pro-
and other proteins, this activity cuts the nascent RNA karyotic and eukaryotic organisms. Some RNA heli-
transcript before addition of the polyA tail by polyA cases work exclusively on RNA. Others can work on
polymerase. The CPSF complex may also play a role DNA:RNA heteroduplexes and DNA substrates. Another
in alternative splicing.80 Exoribonucleases digest single- activity of these enzymes is in the removal of proteins
stranded RNA from the 3′ or 5′ ends. This group from RNA–protein complexes.
of enzymes includes polynucleotide phosphorylase
(PNPase), RNase PH, RNase II, RNase R, RNase D,
RNase T, and others. Like the endoribonuclease RNase PROTEINS AND THE GENETIC CODE
P, RNase D is involved in the 3′ to 5′ processing of
pre-tRNAs. Proteins are the products of transcription and transla-
RNases are ubiquitous, stable enzymes that degrade tion of the nucleic acids. Even though nucleic acids are
all types of RNA. Some RNases are secreted by higher most often the focus of “molecular analysis,” the ulti-
eukaryotes, possibly as an antimicrobial defense mech- mate effect of the information stored and delivered by
anism.81,82 They are of particular concern in laboratories the nucleic acid is manifested in proteins. In the medical
where RNA work is performed because they are very laboratory, analysis of the amount and mutational status
resistant to inactivation. Special precautions are used to of specific proteins has long been performed in situ using
protect RNA from degradation by these enzymes (see immunohistochemistry, on live cells using flow cytome-
Chapter 15, “Quality Assurance and Quality Control in try, and on isolated proteins by enzyme-linked immuno-
the Molecular Laboratory”). sorbent assays, capillary electrophoresis, and western
blots. More recently, global protein analysis by mass
spectrometry (proteomics) has also been applied to clin-
RNA Helicases
ical work. Even if proteins are not being tested directly,
RNA synthesis and processing require the activity of they manifest the phenotype directed by the nucleic acid
helicases to catalyze the unwinding of double-stranded information. In order to interpret the results of nucleic
acid analysis accurately, therefore, it is important to (positive and negative charges) of the side chains of its
understand the movement of genetic information from constituent amino acids.
DNA to protein as dictated by the genetic code. The amino and carboxyl terminal groups of the amino
acids are joined in a carbon-carbon-nitrogen (–C–C–N–)
substituted amide linkage (peptide bond) to form the
Amino Acids
protein backbone (Fig. 1.35). Two amino acids joined
Proteins are polymers of amino acids. Each amino acid together by a peptide bond make a dipeptide. Peptides
has characteristic biochemical properties determined with additional units are tri-, tetra-, pentapeptides, and
by the nature of its amino acid side chain (Fig. 1.32). so forth, depending on how many units are attached to
Amino acids are grouped according to their polarity each other. At one end of the peptide will be an amino
(tendency to interact with water at pH 7) as follows: group (the amino-terminal, or NH2 end), and at the
nonpolar, uncharged polar, negatively charged polar, opposite terminus of the peptide will be a carboxyl
and positively charged polar (Table 1.7). group (the carboxy-terminal, or COOH end). Like the
The properties of amino acids that make up a protein 5′ to 3′ direction of nucleic acids, peptide chains grow
determine the shape and biochemical nature of the from the amino to the carboxy terminus.
protein. A single protein can have separate domains Proteins are polypeptides that can reach sizes of
with different properties. For example, transmembrane more than a thousand amino acids in length. The infor-
proteins have several stretches of hydrophobic amino mation stored in the sequence of nucleotides in DNA is
acids positioned in the lipid membrane of the cell, and transcribed and translated into an amino acid sequence
they might also have hydrophilic or charged extracellu- that will ultimately bring about the genetically coded
lar domains (Fig. 1.33). phenotype.
Amino acids are synthesized in vivo by stereo- Proteins constitute the most abundant macromole-
specific enzymes so that naturally occurring proteins are cules in cells. The collection of proteins encoded in all
made of amino acids of L-stereochemistry. The central of an organism’s DNA is a proteome. The proteome
asymmetric carbon atom of the amino acid is attached of humans is larger than the genome (collection of all
to a carboxyl group, an amino group, a hydrogen atom, genes), possibly 10 times its size, although the exact
and the side chain. Proline differs from the rest of the number of proteins is difficult to assess.83 This is because
amino acids in that its side chain is cyclic, with the a single gene can give rise to more than one protein
amino group attached to the end carbon of the side chain through alternate splicing and other post-transcriptional/
making a five-carbon ring (see Fig. 1.32). post-translational modifications.
Amino acids are also classified by their biosynthetic
origins or similar structures based on a common bio-
synthetic precursor (Table 1.8). Histidine has a unique
synthetic pathway using metabolites common to purine
nucleotide biosynthesis, which affords the connection of
amino acid synthesis to nucleotide synthesis. Advanced Concepts
At pH 7, most of the carboxyl groups of the amino
acids are ionized, and the amino groups are not. The There are two non-canonical amino acids, pyrroly-
ionization can switch between the amino and carboxyl sine and selenocysteine. Pyrrolysine is found in
groups, making the amino acids zwitterions at physi- Archebacteria. Selenocysteine is a component of
ological pH (Fig. 1.34). At certain pH levels, amino selenoproteins, such as glutathione peroxidase and
acids will become completely positively or negatively formate dehydrogenase. Pyrrolysine is encoded
charged. These are the pK values for each amino acid. by UAG and selenocysteine by UGA where they
At the pH where an amino acid is neutral, its positive are inserted instead of a termination signal. In
and negative charges are in balance. This is the pI value. humans, changes in the translation of UGA can
Each amino acid will have its characteristic pI. The pI of lead to symptoms of selenium deficiency.84
a peptide or protein is determined by the ionization state
Charged R groups
H H H H H
+ + +
H3N
+
C COO– H3N
+
C COO– H3N C COO– H3N C COO– H3N C COO–
CH2 CH2 CH2 CH2 CH2

+
CH2 CH2 COO– CH2 C NH
CH2 CH
CH2 COO–
HC NH
NH
CH2
+
+NH C NH2
3
NH2
Lysine Arginine Aspartic acid Glutamic acid Histidine

(Lys) (Arg) (Asp) (Glu) (His)
Polar R groups
H H H H H H
+ + + + +
H3N C COO– H3N C COO– H3N C COO– H3N C COO– +
H3N C COO– H2N C COO–
CH2OH H C OH CH2 CH2 CH2 H2C CH2

CH3 SH C CH2 CH2
H2N O C
H2N O
Serine Threonine Cysteine Asparagine Glutamine Proline

(Ser) (Thr) (Cys) (Asn) (Gln) (Pro)
Nonpolar R groups
H H H H H H
+ + + +
H3N C COO– H3N C COO– H3N C COO– H3N C COO– H3N
+
C COO– H3N
+
C COO–
H CH3 CH CH2 CH2 H C CH3

H3C CH3 CH2 CH CH2
S H3C CH3 CH3
CH3
Glycine Alanine Valine Methionine Leucine Isoleucine
(Gly) (Ala) (Val) (Met) (Leu) (Ile)
Aromatic R groups
H H H
+ + +
H3N C COO– H3N C COO– H3N C COO–
CH2 CH2 CH2
C CH
NH
OH
Phenylalanine Tyrosine Tryptophan

(Phe) (Tyr) (Trp) FIGURE 1.32 Structures of the 20 amino acids. The side
chains are grouped according to their chemical characteristics.
TABLE 1.7 Classification of Amino Acids Based TABLE 1.8 Amino Acid Biosynthetic Groups
on Polarity of Their Side Chains
Amino
Classification Amino Acid Abbreviations Biosynthetic Group Precursor Acids
Nonpolar Alanine Ala, A α-Ketoglutarate group α-Ketoglutarate Gln, Q

Isoleucine Ile, I Glu, E
Leucine Leu, L Pro, P
Methionine Met, M Arg, R
Phenylalanine Phe, F
Pyruvate group Pyruvate Ala, A
Tryptophan Trp, W
Val, V
Valine Val, V
Leu, L
Polar Asparagine Asn, N
Oxalate group Oxaloacetic acid Asp, D
Cysteine Cys, C
Asn, N
Glutamine Gln, Q
Lys, Q
Glycine Gly, G
Ile, I
Proline Pro, P
Thr, T
Serine Ser, S
Met, M
Threonine Thr, T
Tyrosine Tyr, Y Serine group 3-Phosphoglycerate Gly, G
Ser, S
Negatively Aspartic acid Asp, D
charged (acidic) Cys, C
Glutamic acid Glu, E
Aromatic group Chorismate Phe, F
Positively Arginine Arg, R
(Unique biosynthesis) Trp, W
charged (basic) Histidine His, H
Tyr, Y
Lysine Lys, K
His, H
Outside of cell
Extracellular domains
Cell membrane
Intracellular domains
Transmembrane domains
Inside of cell
FIGURE 1.33 Transmembrane proteins have hydrophobic transmembrane domains and hydrophilic domains exposed to the intra-
cellular and extracellular spaces. The biochemical nature of these domains results from their distinct amino acid compositions.
O O O were determined by labeling with 1-fluoro-2,4-

R pK1 R pK2 R dinitrobenzene and digestion with carboxypepti-
OH O– O– dase, respectively. The complete protein was then
+ +
NH3 NH3 NH2
fragmented selectively at lysines and arginines
FIGURE 1.34 An amino acid is positively charged at pK1 with trypsin to 10 to 15 amino acid peptides.
and negatively charged at pK2. At the pH where the positive Fourth, Edmund degradation with phenylisothio-
and negative charges balance (pI), the molecule is neutral. cyanate and dilute acid labeled and removed the
amino terminal residue. This was repeated on
Amino acid 1 Amino acid 2 the same peptide until all the amino acids were
H O identified. Fifth, after another selected cleavage
H R1 of the original protein using cyanogen bromide,
C O N C H
H3N
+
C H + H C O chymotrypsin, or pepsin and identification of the
H R2 peptides by chromatography, the peptides were
O
again sequenced using the Edmund degradation.
Sixth, the complete amino acid sequence could
H2O
be assembled by identification of overlapping
R1
regions.
H H O
+ C
H3N C N C H
C O
O H R2 The sequence of amino acids in a protein determines the
Peptide bond nature and activity of that protein. This sequence is the
primary structure of the protein and is read by con-
+ amino acid 3 vention from the amino terminal end to the carboxy ter-
minal end. Minor changes in primary structure can alter
the activity of proteins dramatically. The single amino
H2O
acid substitution that produces hemoglobin S in sickle
cell anemia is a well-known example. Replacement of a
H R1 H O H R3 soluble glutamine residue with a hydrophobic valine at
+ C C O the sixth residue changes the nature of the protein so that
H3N C N C N C H
C it packs aberrantly in corpuscles and drastically alters
O H R2 H O cell shape. Minor changes in primary structure can have
such drastic effects because the amino acids must often
FIGURE 1.35 The peptide bond is a covalent linkage of the
carboxyl C of one amino acid with the amino N of the next
cooperate with one another to bring about protein struc-
amino acid. One molecule of water is released in the reaction. ture and function.
Advanced Concepts
The primary sequence of proteins can be deter-
mined by a method first described in Fred Sanger ’s Even small peptides can have biological activity.
report of the amino acid sequence of insulin.85,86 Hormones such as insulin, glucagons, corticotro-
This procedure was carried out in six steps. First, pin, oxytocin, bradykinin, and thyrotropin are
the protein was dissociated into amino acids. The examples of peptides (9 to 40 amino acids long)
dissociation products were separated by ion-ex- with strong biological activity. Several antibiot-
change chromatography in order to determine the ics, such as penicillin and streptomycin, are also
type and amount of each amino acid. Second, the peptides.
amino terminal and carboxy terminal amino acids
Advanced Concepts Advanced Concepts

Protein sequence can be inferred from DNA Specialized secondary structures can identify func-
sequence, although the degeneracy of the genetic tions of proteins. Zinc finger motifs are domains
code will result in several possible protein frequently found in proteins that bind to DNA.93
sequences for a given DNA sequence. Many data- These structures consist of two beta sheets followed
bases on the frequency of codon usage in various by an alpha helix with a stabilizing zinc atom.
organisms are available.87,88 These can be used There are three types of zinc fingers, depending on
to more accurately predict amino acid sequences the arrangement of cysteine residues in the protein
from DNA sequence data. sequence. Another example of specialized second-
ary structure is the leucine zipper, also found in
transcription factors.94 The conserved sequence
has a leucine or other hydrophobic residue at each
seventh position for approximately 30 amino acids
Interactions between amino acid side chains fold a arranged in an alpha-helical conformation such
protein into predictable configurations. These include that the leucine side chains radiate outwardly to
ordered beta or beta-pleated sheets and less-ordered facilitate association with other peptides of similar
alpha helices, or random coils. The alpha-helix and structure. Because other amino acids besides
beta-sheet structures in proteins (Fig. 1.36) were leucine can participate in this interaction, the term
first described by Linus Pauling and Robert Corey in basic zipper, or bZip, has been used to describe this
1951.89-92 This level of organization is the secondary type of protein structure. Another similar structure
structure of the protein. Some proteins, especially struc- found in transcriptional regulators is the helix-loop
tural proteins, consist almost entirely of alpha helices or helix,95 consisting of basic amino acids that bind
beta sheets. Globular proteins have varying amounts of consensus DNA sequences (CANNTG) of target
alpha helices and beta sheets. genes. This structure is sometimes confused with
the helix-turn helix. The helix-turn helix is two
alpha helices connected by a short sequence of
amino acids, a structure that can easily fit into the
major groove of DNA.
The Sp1 protein, a eukaryotic transcription regulator, con-

tains a zinc finger motif. The side chains of histidine (H) and
cysteine (C)—part of the zinc finger amino acid sequence,
C-X2-4-C-X3-F-X5-L-X2-H-X3-H (~23 amino acids)—bind a Zn
atom in the active protein. Sp1 binds DNA in the regulatory
region of genes. Note: For the single-letter amino acid code, see
Tables 1.7 and 1.8. X in this consensus denotes any amino acid.
The lambda repressor, a transcription factor of the bacterio-

phage lambda, has a helix-turn-helix motif. One of each of the
helices fits into the major groove of the DNA. Lambda repres-
sor prevents transcription of genes necessary for active growth
of the bacteriophage, leading to host-cell lysis.
A α Helix B β Pleated sheet
FIGURE 1.36 Secondary structure of proteins includes the

alpha helix (A) and the beta pleated sheet (B). The ribbon-like The secondary structures of proteins are further folded
structures in the pictures are composed of chains of amino and arranged into a tertiary structure. Tertiary struc-
acids hydrogen-bonded through their side chains. ture is also important for protein function. If a protein
loses its tertiary structure, it is denatured. Mutations in Regulatory Structural Regulatory

sequence Promoter sequence sequence
DNA that substitute different amino acids in the primary 5′ PO OH 3′
structure can also alter tertiary structure. Denatured 3′ HO OP 5′
or improperly folded proteins are not functional. Pro- DNA
teins can also be denatured by heat (e.g., the albumin FIGURE 1.37 A gene contains not only structural (coding)
in egg white) or by conformations forced on innocuous sequences but also sequences important for regulated tran-
peptides by infectious prions. Aggregations of prion- scription of the gene. These include the promoter, where RNA
induced aberrantly folded proteins cause transmissible polymerase binds to begin transcription, and regulatory
spongiform encephalopathies, such as Creutzfeldt–Jakob regions, where transcription factors and other regulatory
disease and bovine spongiform encephalitis (mad cow factors bind to stimulate or inhibit transcription by RNA
polymerase.
disease).
Two proteins bound together to function form a
dimer, three form a trimer, and four form a tetramer. Genes were first studied by tracking mutations (changes
Proteins that work together in this way are called oligo- in the order or sequence of nucleotides in the DNA) that
mers, each component protein being a monomer. This took away their function and observing the resulting
is the quaternary structure of proteins. The combi- phenotype. A gene was considered that part of a chro-
natorial nature of protein function may account for the mosome responsible for the phenotype affected by muta-
genetic complexity of higher organisms without a con- tion. Genes were not delineated well in terms of their
current increase in gene number. physical size but were mapped relative to each other
Proteins are classified according to function as based on the frequency of recombination between them.
enzymes and as transport, storage, motility, structural, A gene contains not only structural sequences that
defense, or regulatory proteins. Enzymes and transport, code for an amino acid sequence but also regulatory
defense, and regulatory proteins are usually globular sequences that are important for the regulated expres-
in nature, making them soluble and allowing them to sion of the gene (Fig. 1.37). Cells expend a good deal
diffuse freely across membranes. Structural and motility of energy to coordinate protein synthesis so that the
proteins are fibrous and insoluble. proper proteins are available at specific times and in
In contrast to simple proteins that have no other com- specific amounts. Loss of this controlled expression will
ponents except amino acids, conjugated proteins do result in an abnormal phenotype, even though there may
have components other than amino acids. The nonpro- be no changes in the structural sequence of the gene.
tein component of a conjugated protein is the nonpro- The failure to appreciate the importance of proximal
tein prosthetic group. Examples of conjugated proteins and distal regulatory sequences was another source of
are those covalently attached to lipids (lipoproteins), for confusion in early efforts to define a gene. Regulatory
example, low-density lipoproteins; sugars (glycopro- effects and the interaction between proteins still chal-
teins), for example, mucin in saliva; and metal atoms lenge the interpretation of genetic analyses in the clin-
(metalloproteins), for example, ferritin. One of the most ical laboratory.
familiar examples of a conjugated protein is hemoglobin.
Hemoglobin is a tetramer, having four Fe2+-containing
The Genetic Code
heme groups, one covalently attached to each monomer.
The nature of a gene was further clarified with the deci-
phering of the genetic code by Francis Crick, Marshall
Genes
Nirenberg, Philip Leder, Gobind Khorana, and Sydney
A gene is defined as the ordered sequence of nucleo- Brenner.40,96-99 The genetic code is not information in
tides on a chromosome that encodes a specific func- itself but is a dictionary to translate the 4-nucleotide
tional product. A gene is the fundamental physical and sequence information in DNA to the 20–amino acid
functional unit of inheritance. The physical definition sequence information in proteins.
of a gene was complicated in early studies because of The triplet nature of the genetic code was surmised
the methods used to define units of genetic inheritance. based on mathematical considerations. It was reasoned
that the smallest set of four possible letters that would The challenge was to decipher this triplet code and
yield enough unique groups to denote 20 different amino prove its function. The simplest way to prove the code
acids was three. A 1-nucleotide code could only account would have been to determine the order of nucleotides
for 41 = 4 different amino acids, whereas a 2-nucleotide in a stretch of DNA coding for a protein and compare
code would yield 42 = 16 different possibilities. A it with the order of amino acids in the protein. In the
3-nucleotide code would give 43 = 64 different possibili- early 1960s, protein sequencing was possible, but only
ties, enough to account for all 20 amino acids. limited DNA sequencing was available. Marshall Niren-
berg made the initial attempts at the code by using short
synthetic pieces of RNA to support protein synthesis
Histooricaal Higghlligghtts in a cell-free extract of E. coli. In each of 20 tubes, he
placed a different radioactive amino acid, then added
In the early 1960s, Seymour Benzer used the cell lysate from E. coli and an RNA template. In the
T4 bacteriophage to investigate the gene more first definitive experiment, the input RNA template was
closely. By mixing the phage with several differ- a polymer of uracil, UUUUUUU. . . . If the input tem-
ent phenotypes, he could observe the restoration plate supported the synthesis of protein, the radioactive
of normal phenotype (complementation) of one amino acids would be joined together, and the radioac-
mutant by introducing a phage with a different tivity would be detected in a precipitable protein. On
mutation. Benzer could distinguish mutations May 27, 1961, Nirenberg measured radioactive protein
that could complement each other, even though levels from 19 of the 20 vials at around 70 counts/mg;
they affected the same phenotype and mapped to however, one vial, which contained the amino acid phe-
the same location in the phage DNA. He deter- nylalanine, yielded protein of 38,000 counts/mg. If the
mined that these were mutations in different 3-nucleotide code was correct, then UUU was the codon
places within the same gene. He organized many for phenylalanine.
mutants into a series of sites along the linear array After the first successful demonstration of this strat-
of the phage chromosome so that he could struc- egy, other templates were tested. Each synthetic nucleic
turally define the gene as a continuous linear span acid incorporated different amino acids, based on the
of genetic material. composition of bases in the RNA sequence. Codes for
phenylalanine (UUU), proline (CCC), lysine (AAA), and
glycine (GGG) were soon deduced from the translation
of RNA synthesized from a single nucleotide population.
Histooricaal Higghlligghtts More of the code was indirectly deduced using mixtures
of nucleotides at different proportions. For instance,
The interesting history of the breaking of the an RNA molecule synthesized from a 2:1 mixture of
genetic code began with a competitive scram- U and C polymerized mostly phenylalanine and leucine
ble. A physicist and astronomer, George Gamow, into protein. Similar tests with other nucleotide mix-
organized a group of scientists to concentrate on tures resulted in distinct amino acid incorporations.
the problem. They called themselves the RNA Although the nucleotide content of each RNA molecule
Tie Club. Each of the 20 members wore a tie in these tests was known, the exact order of nucleotides
emblazoned with a depiction of RNA and a pin in the triplet was not known, leading to inconclusive
depicting a different amino acid. The group met results.
regularly during the 1950s. They made some pro- Nirenberg and Leder used another technique to get
gress, including Watson and Crick’s “adaptor” at the basic structure of the code. They observed the
hypothesis (predicting tRNA) and Gamow’s binding of specific amino acids to three-base RNA mol-
mathematical prediction of three nucleotides ecules (triplets) in ribosome–tRNA mixtures. By noting
coding for one amino acid. Ultimately, however, which triplet/amino acid combination resulted in binding
they did not exclusively break the genetic code. of the amino acid to ribosomes, they were able to assign
50 of the 64 possible triplets to specific amino acids.
Meanwhile, Gobind Khorana had developed another carrier tRNA and the mRNA template during protein
system. He synthesized longer RNA polymers of known translation. Wobble can also be observed in the third
nucleotide sequence. With polynucleotides of repeated position of the anticodon as well as the codon.101 Codon
sequence, he could predict and then observe the pep- selection may be important for the differentiation of
tides that would come from that RNA. For example, a human tissues and may also have a role in the develop-
polymer consisting of two bases, such as . . . UCUCU- ment of diseases, such as cancer, where differentiation
CUCUCUCUC . . ., was expected to code for a peptide pathways are altered.102 Thus, all amino acids except
of two different amino acids, one coded for by UCU and leucine, serine, and arginine are selected by the first two
one by CUC. This polymer yielded a peptide with the letters of the genetic code. The first two letters, however,
sequence . . . Ser-Leu-Ser-Leu. . . . This experiment did do not always specify unique amino acids. For example,
not indicate which triplet coded for which amino acid, CA begins the code for both histidine and glutamine.
but combined with the results from Nirenberg and Leder, Three codons—UAG, UAA, and UGA—that terminate
the UCU was assigned to serine and CUC to leucine. protein synthesis are termed nonsense codons. UAG,
By 1965, all 64 triplets, or codons, were assigned to UAA, and UGA were named amber, ocher, and opal,
amino acids (Fig. 1.38). Once the code was confirmed, respectively, when they were first defined in bacterial
specific characteristics of it were apparent. The code is viruses.
redundant, such that all but two amino acids (methionine The characteristics of the genetic code have conse-
and tryptophan) have more than one codon. Triplets quences for molecular analysis. Mutations or changes in
coding for the same amino acid are similar, often dif- the DNA sequence will have different effects on pheno-
fering in the third base of the triplet. Crick first referred type, depending on the resultant changes in the amino
to this as wobble in the third position.100 Wobble is also acid sequence. Accordingly, mutations range from phe-
used to describe the movement of the base in the third notypically silent to drastic. This will be discussed in
position of the triplet to form novel pairing between the more detail in later chapters.
Second position of codon

U C A G
UUU UAU UGU U
UCU Tyrosine
UUC Phenylalanine UCC UAC UGC Cysteine C
U Serine
UUA UCA UAA Ter (end) UGA Ter (end) A
Leucine UCG
UUG UAG Ter (end) UGG Tryptophan G
CAU U
CUU CCU Histidine CGU
CUC CCC CAC CGC C
C Leucine Proline Arginine Third position
First position
CUA CCA CAA CGA A

CUG CCG CAG Glutamine CGG
G
AUU AAU AGU U
ACU Asparagine Serine
AUC Isoleucine ACC AAC AGC C
A AUA Threonine
ACA AAA AGA A
AUG Methionine ACG AAG Lysine AGG Arginine G
GAU Aspartic U
GUU GCU GGU
GUC GCC GAC acid GGC C
G Valine Alanine Glycine
GUA GCA GAA Glutamic GGA A
GUG GCG GAG GGG
acid G
FIGURE 1.38 The genetic code. Codons are read as the nucleotide in the left column, then the row at the top, and then the right
column. Note how there are up to six codons for a single amino acid. Only methionine and tryptophan have a single codon. Note
also the three termination codons (ter): UAA, UAG, UGA.
An interesting observation about the genetic code is charging, a reaction catalyzed by 20 aminoacyl tRNA
that, with limited exceptions, the repertoire of amino synthetases. The Mg++-dependent charging reaction
acids is limited to 20 in all organisms, regardless of was first described by Hoagland and Zamecnik, who
growing environments. Thermophilic and cryophilic observed that amino acids incubated with ATP and
organisms adapt to growth at 100°C and freezing tem- the cytosol fraction of liver cells became attached to
peratures, respectively, not by using structurally differ- heat-soluble RNA (tRNA).108 The reaction takes place
ent amino acids but by varying the combinations of the in two steps. First, the amino acid is activated by the
naturally occurring amino acids. Cells have strict control addition of AMP:
and editing systems to protect the genetic code and avoid amino acid + ATP → aminoacyl-AMP + PPi
incorporation of unnatural amino acids into proteins.
Second, the activated amino acid is joined to the tRNA:
Studies have shown that it is possible to manipulate the
genetic code to incorporate modified amino acids.103,104 aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP
This ability to introduce chemically or physically reac- The product of the reaction is an ester bond between the
tive sites into proteins in vivo has significant implica- 3′ hydroxyl of the terminal adenine of the tRNA and the
tions in biotechnology. carboxyl group of the amino acid.
Advanced Concepts
In humans, the termination codon UGA also codes Advanced Concepts
for selenocysteine. Selenoproteins have UGA
codons in the middle of their coding regions. In According to evolutionary theory, the genetic code
the absence of selenium, protein synthesis stops has evolved over millions of years of selection. An
prematurely in these genes. interesting analysis compared the natural genetic
code shared by all living organisms with mil-
lions of other possible triplet codes generated by
computer (four nucleotides coding for 20 amino
TRANSLATION acids).109 The results showed that the natural code
was significantly more resistant to damaging
Amino Acid Charging
changes (mutations in the DNA sequence) com-
After transcription of the sequence information in DNA pared with the other possible codes. The code is
to RNA, the transcribed sequence must be transferred still undergoing “fine-tuning” through common
into proteins. Through the genetic code, a specific mechanisms in prokaryotes and eukaryotes.110
nucleic acid sequence is translated to an amino acid
sequence and, ultimately, to a phenotype. As proposed in
the adaptor hypothesis, there must be a molecular factor
that can recognize components of both nucleic acid and
protein sequences. This factor is transfer RNA (tRNA). Advanced Concepts
Within the 75- to 95-ribonucleotide sequence of each
tRNA is a three-base anticodon, complementary to the Once the amino acid is esterified to the tRNA, it
codon of a specific amino acid. With the redundancy of makes no difference in the specificity of its addi-
the genetic code, there are over 50 tRNAs in humans and tion to the protein. The fidelity of translation is now
40 in bacteria.105,106 There are also tRNAs with the same determined by the anticodon of the tRNA as an
anticodon but with a sequence outside of the anticodon adaptor between mRNA and the growing protein.
or tRNA isodecoders, which are expressed differently This association has been exploited by attach-
in different cells and different stages of development.107 ing amino acids synthetically to selected tRNAs.
Protein synthesis starts with activation of the amino If amino acids are attached to tRNAs carrying
acids by covalent attachment to tRNA, or tRNA
Protein Synthesis
anticodons to UAG, UAA, or UGA, the peptide
chain will continue to grow instead of terminating Translation takes place on ribosomes, ribonucleoprotein
at the stop codon. These tRNAs are called sup- particles first observed by electron microscopy of animal
pressor tRNAs because they can suppress point cells. In the early 1950s, Paul Zamecnik demonstrated
mutations (single-base-pair changes) that gener- that these particles were the site of protein synthesis in
ate stop codons within a protein-coding sequence. bacteria.112 There can be as many as 10 million ribo-
Suppression of premature termination mutations somes in a eukaryotic cell, depending on cell type, and
in mutated genes has been suggested as a type of over 70,000 ribosomes in a bacterial cell, depending on
gene therapy for mutation-driven diseases.111 the growth rate of the cell.
Ribosomal structure is similar in prokaryotes and
eukaryotes (see Fig. 1.27). In prokaryotes, 70S ribo-
somes are assembled from a 30S small subunit and a
There are 20 amino acid tRNA synthetase enzymes, one 50S large subunit, in association with mRNA and initiat-
for each amino acid. Designated as class I and class II ing factors. (S stands for sedimentation units in density-
synthetases, these enzymes interact, respectively, with gradient centrifugation, a method used to determine
the minor or major groove of the tRNA acceptor arm. the sizes of proteins and protein complexes.) The 30S
Both classes also recognize tRNAs by their anticodon subunit (1 million daltons) is composed of a 16S ribo-
sequences and amino acids by their side chains. Only the somal RNA (rRNA) and 21 ribosomal proteins. The
appropriate tRNA and amino acid will fit into its cognate 50S subunit (1.8 million daltons) is composed of a
synthetase (Fig. 1.39). An errant amino acid bound to the 5S rRNA, a 23S rRNA, and 34 ribosomal proteins.
wrong synthetase will dissociate rapidly before any con- Eukaryotic ribosomes are slightly larger (80S) and more
formation changes and charging can occur. In another complex, with a 40S small subunit (1.3 million daltons)
level of editing, mischarged aminoacylated tRNAs are and a 60S subunit (2.7 million daltons). The 40S subunit
hydrolyzed at the point of release from the enzyme. is made up of an 18S rRNA and about 30 ribosomal
proteins. The 60S subunit contains a 5S rRNA, a 5.8S
rRNA, a 28S rRNA, and about 40 ribosomal proteins.
Protein synthesis in the ribosome almost always
Aminoacyl-tRNA synthetase starts with the amino acid methionine in eukaryotes
and N-formylmethionine in bacteria, mitochondria, and
tRNA chloroplasts. Initiating factors that participate in the for-
Ile mation of the ribosome complex differentiate the initi-
Val
ating methionyl tRNAs from those that add methionine
internally to the protein.113 In protein translation, the
small ribosomal subunit first binds to initiation factor
Gln 3 (IF-3) and then to specific sequences near the 5′ end
of the mRNA, the ribosomal binding site. This guides
Phe
the AUG codon (the “start” codon) to the proper place
in the ribosomal subunit. Another initiation factor, IF-2
bound to GTP and the initiating tRNAMet or tRNAfMet,
Thr then joins the complex (Fig. 1.40). The large ribosomal
subunit then associates with the hydrolysis of GTP and
release of GDP and phosphate, IF-2, and IF-3. The
resulting functional 70S or 80S ribosome is the initia-
FIGURE 1.39 Five aminoacyl tRNA synthetases. Each tion complex. In this complex, the tRNAMet or tRNAfMet
enzyme is unique for a tRNA and its matching amino acid. The is situated in the peptidyl site (P site) of the functional
specificity of these enzymes is key to the fidelity of translation. ribosome. tRNAMet or tRNAfMet can only bind to the
Amino
acid
Ribosomal subunits
tRNA
5′
mRNA 3′
Components
Initiation
Recycling
5′ Amino acid
3′
tRNA
Elongation
Polypeptide Termination
5′ 5′
3′ 3′
Polypeptides More ribosomes

First ribosome attach to the 5′
reaches termination end of mRNA
codon
5′ 3′
Polyribosomal complex
FIGURE 1.40 Assembly of the small ribosome subunit with mRNA and then the large ribosomal subunit; charged tRNA initiates
RNA synthesis (initiation). Binding of charged tRNAs and formation of the peptide bond produce the growing polypeptide (elon-
gation). Several ribosomes can simultaneously read a single mRNA (polyribosome complex). When the complex encounters a
nonsense codon, protein synthesis stops (termination), and the components are recycled.
P site in the ribosome, which is formed in combination Growing peptide tRNAleu

by both ribosomal subunits. In contrast, all other tRNAs Amino
tRNAser acid
bind to an adjacent site, the aminoacyl site (A site) of
tRNAtyr
the ribosome.
Synthesis proceeds in the elongation step where the P site
A A C
tRNA carrying the next amino acid binds to the A site A site
G
of the ribosome in a complex with elongation factor Tu A G
(EF-Tu) and GTP (Fig. 1.41). The fit of the incoming A U A
C CG AU C C U A U U UGU U C CG A GU
tRNA takes place by recognition and then proofread-
5′ 3′
ing of the codon–anticodon base pairing. Hydrolysis of mRNA
GTP by EF-Tu occurs between these two steps.114 The Ribosome
EF-Tu-GDP is released, and the EF-Tu-GTP is regener-
ated by another elongation factor, EF-Ts. Although these
interactions ensure the accurate pairing of the first two
codon positions, the pairing at the third position is not Translocation
as stringent, which might be related to the wobble in
the genetic code.
tRNAleu
The first peptide bond is formed between the tRNAtyr
amino acids in the A and P sites by transfer of the
N-formylmethionyl group of the first amino acid to A U A A A C
C CG AU C C U A U U UGU U C CG A GU
the amino group of the second amino acid, generating 5′ 3′
a dipeptidyl-tRNA in the A site. This step is catalyzed
by an enzymatic activity in the large subunit, peptidyl
tRNAphe
transferase. This activity is mediated through RNA, but
proteins in the vicinity of the active site of the pepti- tRNAtyr
dyl transferase center of the ribosome may influence its
organization or the efficiency of the reaction.115,116 After tRNAleu
formation of the peptide bond, the ribosome moves, A A G
shifting the dipeptidyl-tRNA from the A site to the P site A
with the release of the “empty” tRNA from a third posi- A U
tion, the E site, of the ribosome. This movement (trans- A A C

location) of tRNAs across a distance of 20 angstroms C CG AU C C U A U U UGU U C CG A GU
from the A to the P site and 28 angstroms from the P to 5′ 3′
the E site requires elongation factor EF-G. As the ribo-

somal complex moves along the mRNA, the growing
FIGURE 1.41 Incoming charged tRNAs bind to the A site of
peptide chain is always attached to the incoming amino the ribosome, guided by matching codon–anticodon pairing.
acid. Two GTPs are hydrolyzed to GDP with the addi- After formation of the peptide bond between the incoming
tion of each amino acid. This energy-dependent trans- amino acid and the growing peptide, the ribosome moves to
location occurs with shifting and rotation of ribosomal the next codon in the mRNA, translocating the peptide to the
subunits (Fig. 1.42). P site and creating another A site for the next tRNA.
During translation, the growing polypeptide begins
to fold into its mature conformation. This process is into mature protein. In the absence of this activity, unfin-
assisted by molecular chaperones.117 These specialized ished proteins might bind to each other and form non-
proteins bind to the large ribosomal subunit, forming a functional aggregates. Termination of the amino acid
hydrophobic pocket that holds the emerging polypeptide chain is signaled by one of the three nonsense, or ter-
(Fig. 1.43). Chaperones apparently protect the growing mination, codons—UAA, UAG, or UGA—which are
unfinished polypeptides until they can be safely folded not charged with an amino acid. When the ribosome
Ribosome
P site A site
A2451
:
Peptide O H R
:N O
O
H O
tRNA
P site tRNA
A site
O R
Peptide
O
H N
O H O
tRNA tRNA
FIGURE 1.42 Protein synthesis (translation) as it takes place in the ribosome. The peptide bond is formed in an area between the
large and small subunits of the ribosome. The ribozyme theory holds that the ribosome is an enzyme that functions through RNA
and not protein. The close proximity of only RNA to this site is evidence for the ribozyme theory.
Ribosome
3′ 3′ 3′
5′ 5′ 5′
mRNA
Growing polypeptide
Chaperone
Folded
peptide
FIGURE 1.43 Molecular chaperones catch the growing peptide as it emerges from the active site. The peptide goes through
stages of holding (left), folding (center), and release (right). When the protein is completely synthesized and released from the
ribosome, it should be in its folded state. This protects the nascent (growing) peptide from harmful interactions with other proteins
in the cell before it has had an opportunity to form its protective and active tertiary structure.
encounters a termination codon, termination, or release

factors (R1, R2, and S in E. coli), will cause hydrolysis STUDY QUESTIONS
of the finished polypeptide from the final tRNA, release
of that tRNA from the ribosome, and dissociation of the DNA Structure and Function
large and small ribosomal subunits. In eukaryotes, ter-
mination codon–mediated binding of polypeptide chain 1. What is the function of DNA in the cell?
release factors (eRF1 and eRF3) triggers hydrolysis of
peptidyl-tRNA at the ribosomal peptidyl transferase 2. Compare the structure of the nitrogen bases. How do
center.118,119 E. coli can synthesize a 300- to 400-amino purines and pyrimidines differ?
acid protein in 10 to 20 seconds. Because the protein
takes on its secondary structure as it is being synthe- 3. Write the complementary sequence to the
sized, it already has its final conformation when it is following:
released from the ribosome.
5′ AGGTCACGTCTAGCTAGCTAGA3′
4. Which of the ribose carbons participate in the

Advanced Concepts phosphodiester bond?
In eukaryotes, accumulation of unfolded proteins 5. Which of the ribose carbons carries the nitrogen
in the endoplasmic reticulum elicits an unfolded base?
protein response (ER stress), intended to correct
the problem by cessation of protein synthesis 6. Why does DNA polymerase require primase
or induction of more chaperones. If correction activity?
doesn’t occur, ER stress will send the cell into a
programmed death (apoptosis). ER stress has been Restriction Enzyme Analysis
found to be associated with a number of diseases,
including diabetes, neurodegenerative disorders, 1. A plasmid was digested with the enzyme HpaII.
cancer, and cardiovascular disease.120 On agarose gel electrophoresis, you observe three
bands, 100, 230, and 500 bp.
a. How many HpaII sites are present in this
plasmid?
In bacteria, translation and transcription occur simul- b. What are the distances between each site?
taneously. In nucleated cells, the majority of transla- c. What is the size of the plasmid?
tion occurs in the cytoplasm. Several lines of evidence d. Draw a picture of the plasmid with the HpaII
suggest, however, that some translation might also occur sites.
in the nucleus. One line of evidence is that nuclei contain
A second cut of the plasmid with BamH1 yields two
factors required for translation. Furthermore, isolated
pieces, 80 and x bp.
nuclei can aminoacylate tRNAs and incorporate amino
acids into proteins. e. How many BamH1 sites are in the plasmid?
A cellular surveillance system for RNA with pre- f. What is x in base pairs (bp)?
mature termination codons, nonsense-mediated decay
(NMD),121 a degradation of messenger RNAs with 2. How would you determine where the BamH1 sites
premature termination codons, supposedly occurred in are in relation to the HpaII sites?
mammalian nuclei. Further investigations have shown,
however, that NMD may not occur in the nuclei of lower 3. The plasmid has one EcoR1 site into which you
eukaryotes. want to clone a blunt-ended fragment. What type
of enzyme could turn an EcoR1 sticky end into Proteins and the Genetic Code
a blunt end?
1. Indicate whether the following peptides are
Recombination and DNA Transfer hydrophilic or hydrophobic.
a. MLWILAA
1. Describe how DNA moves from cell to cell b. VAIKVLIL
by (a) conjugation, (b) transduction, and c. CSKEGCPN
(c) transformation. d. SSIQKNET
e. YAQKFQGRT
2. Which of the three interactions in question 1 would f. AAPLIWWA
be prevented by a barrier between two mating g. SLKSSTGGQ
strains that stops bacterial cells, but not smaller
particles? 2. Is the following peptide positively or negatively
charged at neutral pH?
3. After meiosis, gametes produced from diploid
organisms are ___________ (haploid/diploid). GWWMNKCHAGHLNGVYYQGGTY
3. Consider an RNA template made from a

RNA Secondary Structure 2:1 mixture of C:A. What would be the three
amino acids most frequently incorporated
1. Draw the secondary structure of the following RNA. into protein?
The complementary sequences (inverted repeat) are
underlined. 4. What is the peptide sequence encoded in
AUAUAUAUAUAUAUA . . .?
5′CAUGUUCAGCUCAUGUGAACGCU 3′
5. Write the anticodons 5′ to 3′ of the following
2. Underline two inverted repeats in the following amino acids:
RNA.
a. L
5′CUGAACUUCAGUCAAGCAAAGAGUUUGCACUG3′
b. T
c. M
d. H
RNA Processing e. R
f. I
1. Name three processing steps undergone by
messenger RNA. 6. A protein contains the sequence
LGEKKWCLRVNPKGLDESKDYLSLKSKYLLL.
2. What is the function of polyadenylate polymerase? What is the likely function of this protein? (Note:
See Advanced Concepts box.)
3. What is unusual about the phosphodiester bond
formed between mRNA and its 5′ cap? 7. A histone-like protein contains the sequence
PKKGSKKAVTKVQKKDGKKRKRSRK. What
4. The parts of the hnRNA that are translated into characteristic of this sequence makes it likely to
protein (open reading frames) are the __________. associate with DNA?
5. The intervening sequences that interrupt protein- 8. A procedure for digestion of DNA with a
coding sequences in hnRNA are _____________. restriction enzyme includes a final incubation step
of 5 minutes at 95°C. What is the likely purpose of 16. Pandey M, Syed S, Donmez I, Patel G, Ha T, Patel SS. Coordi-
this final step? nating DNA replication by means of priming loop and differen-
tial synthesis rate. Nature 2009;462:940–943.
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following conjugated proteins: Enzymatic synthesis of deoxyribonucleic acid. VII. Synthesis of
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Chapter 2
Gene Expression and Epigenetics
Outline Objectives
TRANSCRIPTION 2.1 Define epigenetics, and list examples of epigenetic
Transcription Initiation phenomena.
Transcription Elongation 2.2 Demonstrate gene regulation using the Lac operon
Transcription Termination as an example.
REGULATION OF TRANSCRIPTION 2.3 Categorize non-coding RNA and its mechanism of
Regulation of Messenger RNA Synthesis at Initiation action.
Post-Transcriptional Regulation 2.4 Describe histone modification and the histone
Post-Translational Regulation code.
EPIGENETICS 2.5 Explain nucleic acid methylation and its effect on
Histone Modification gene expression.
Nucleic Acid Methylation 2.6 Identify post-translational protein modifications.
CLASSIFICATION OF EPIGENETIC FACTORS
NONCODING RNAs
MicroRNAs
Small Interfering RNAs
Other Small RNAs
Long Noncoding RNAs
57
TRANSCRIPTION (phenotype). Heritable changes in phenotype arise from

structural and chemical sequence changes in DNA. More
DNA can only store information. In order for this infor- immediate changes in phenotype are brought about by
mation to be utilized, it must be transcribed and then selective expression (transcription and translation) of
translated into protein, a process called gene expression. genes. Thus, genetics and epigenetics combine to adapt
A specific type of RNA, messenger RNA (mRNA). cells and organisms to their environment.
carries the information in DNA to the ribosomes, where
it is translated into protein. Transcription Initiation
Transcription is the copying of one strand of DNA
into RNA by a process similar to that of DNA replication. RNA polymerase and its supporting accessory proteins
This activity, catalyzed by RNA polymerase, occurs assemble at a start site in DNA, a specific sequence of
mostly in interphase. A single type of RNA polymerase bases called the promoter. Sites for initiation of tran-
catalyzes the synthesis of all RNA in most prokaryotes. scription (RNA synthesis) greatly outnumber DNA ini-
There are three types of RNA polymerases in eukary- tiation sites (origins of replication) in both prokaryotes
otes: RNA polymerase pol I, pol II, and pol III. Pol I and eukaryotes. There are also many more molecules
and III synthesize noncoding RNA.1 Pol II is responsible of RNA polymerase than DNA polymerase in the cell.
for the synthesis of mRNA, the type of RNA that carries Although functionally catalyzing the same reaction,
genetic information to be translated into protein (Table RNA polymerases in prokaryotes and eukaryotes differ
2.1). Evidence suggests that transcription takes place at and work with different supporting proteins to find and
discrete stations of the nucleus into which the DNA mol- bind to DNA in preparation for transcription. In prokary-
ecules move.2,3 One of these sites, the nucleolus, is the otes, a basal transcription complex comprised of the large
location of ribosomal RNA synthesis (Table 2.2). and small subunits of RNA polymerase and additional
Cells with the same DNA genome (genotype) can sigma factors assembles at the start site. The eukaryotic
have vastly different morphologies and activities transcription complex requires RNA polymerase and up
to 20 additional factors for accurate initiation. Initiation
of RNA synthesis is regulated in all organisms so that
genes are transcribed as required by specific cell types.
TABLE 2.1 RNA Polymerases
Enzyme Template Product Transcription Elongation

RNA polymerases in both eukaryotes and prokaryotes
E. coli RNA polymerase II DNA mRNA
synthesize RNA using the base sequence of one strand
RNA polymerase I DNA rRNA
RNA polymerase II DNA mRNA
RNA polymerase III DNA tRNA, snRNA

TABLE 2.2 Cellular Location and Activity of RNA
Mitochondrial RNA DNA mRNA Pol I, II, and III in Eukaryotes
polymerase
Mammalian DNA DNA primers Type Location Products α-Amanitin

polymerase α
I Nucleolus 18s, 5.8s, 28s Insensitive
HCV RNA polymerase RNA Viral genome rRNA
Dengue virus RNA RNA Viral genome II Nucleus mRNA, snRNA Inhibited
polymerase
III Nucleus tRNA, 5s rRNA Inhibited by high
PolyA polymerase None PolyA tails concentration
Chapter 2 • Gene Expression and Epigenetics 59
mRNA
RNA
A C G
U G polymerase
5′ C
U
G DNA template
A 5′
A
G T G A C A A G T A C C G T A G C T A
A C U G U U C A U G G C A U C
T T
3′ 3′
DNA A
5′ A
C A C T G T T C A T G G C A T C G
DNA complement of template
FIGURE 2.1 RNA transcription proceeds by synthesis of the RNA molecule using one strand of the DNA template. The copied
strand is complementary to the RNA product, whereas the homologous strand has the same sequence as the RNA product.
of the double helix (the antisense strand) as a guide

Transcription Termination
(Fig. 2.1). The sense strand of the DNA template has
a sequence identical to that of the RNA product (except RNA synthesis terminates differently in prokaryotes and
for the U for T substitution in RNA), but it does not eukaryotes. In prokaryotes, RNA synthesis is respon-
serve as the template for the RNA. sive to protein products, such that high levels of a gene
RNA polymerases work more slowly than DNA product induce termination of its own synthesis. This
polymerases (50 to 100 bases/sec for RNA synthesis synthesis is accomplished in some genes by interactions
vs. 1,000 bases/sec for DNA replication) and with less between RNA polymerase and termination signals in the
fidelity. The DNA double helix is locally unwound into DNA template. In other genes, an additional transcription
single strands to allow the assembly and passage of the complex factor, rho, is required for termination. Rho is
transcription machinery, forming a transcription bubble. a helicase enzyme that associates with RNA polymerase
Unlike DNA synthesis, RNA synthesis does not and inactivates the elongation complex at a cytosine-rich
require priming. The first ribonucleoside triphosphate termination site in the DNA.4,5 Rho-independent termi-
retains all of its phosphate groups as the RNA is poly- nation occurs at G:C-rich regions in the DNA, followed
merized in the 5′ to 3′ direction. Subsequent ribonucleo- by A:T-rich regions. The G:C bases are transcribed into
side triphosphates retain only the alpha phosphate, the RNA and fold into a short double-stranded hairpin,
one closest to the ribose sugar. The other two phosphate which slows the elongation complex. The elongation
groups are released as orthophosphate during the syn- complex then dissociates as it reaches the A:T-rich area.
thesis reaction. In eukaryotes, mRNA synthesis, catalyzed by pol II,
After initiation of mRNA synthesis, the 5′ end of the proceeds along the DNA template until a polyadenyla-
growing transcript is covalently attached to a methylated tion signal (polyA site) is encountered. At this point,
guanine residue (7-methylguanosine) in a 5′ to 5′ cova- the process of termination of transcription is activated.
lent bond. Capping occurs in three steps: (1) hydrolysis There is no consensus sequence in DNA that specifies
of the 5′ phosphate from the pre-mRNA, (2) transfer of a the termination of transcription. As the polymerase pro-
guanine monophosphate nucleoside to the 5′ diphosphate ceeds past the polyA site, the nascent mRNA is released
end, and (3) methylation of the attached guanine in the by an endonuclease associated with the carboxy terminal
N7 position. The cap protects the end of the growing end of pol II. RNA synthesized beyond the site trails out
pre-mRNA (or hnRNA) and facilitates ribosome binding of the polymerase and is bound by another exonuclease
and efficient translation. that begins to degrade the RNA 5′ to 3′ toward the RNA
polymerase. When the exonuclease catches up with the

polymerase, transcription stops. Subsequently, polyade- are funneled through a pore in the cleft beneath
nylate polymerase enzymatically adds 20 to 200 adenine the active site (pore 1). In the active site, the DNA
nucleotides to the 3′ end of the new transcripts. Polyad- strands are separated and the RNA chain is elon-
enylation is important for the localization, stability, and gated, driven by cleavage of phosphates from each
translation of mRNA in the cell. Almost all pol II tran- incoming ribonucleoside triphosphate. The result-
scripts are polyadenylated. ing DNA–RNA hybrid moves out of the active site
nearly perpendicular to the DNA coming into the
cleft. After reaching a length of 10 bases, the newly
synthesized RNA dissociates from the hybrid and
Advanced Concepts leaves the complex through an exit channel. Three
protein loops—“rudder,” “lid,” and “zipper”—are
Some pol II transcripts have alternative polyade- involved in hybrid dissolution and exit of the RNA
nylation sites. Termination of the transcriptions at product.7,8
alternative locations on the transcript produces dif-
ferent transcripts (isoforms) from the same gene.6
Pol I terminates transcription just prior to a site
in the DNA (Sal box) with the cooperation of a For some genes whose products are in continual use by
termination factor, TTF1.6 The pol III termination the cell, gene expression is constant or constitutive. For
signal is a run of adenine residues in the template. other genes, expression is tightly regulated throughout
Pol III transcription termination requires a termi- the life of the cell. Because gene products often function
nation factor. together to bring about a specific cellular response, spe-
cific combinations of proteins in stoichiometric balance
are crucial for cell differentiation and development.
Protein availability and function are controlled at the
REGULATION OF TRANSCRIPTION levels of transcription, translation, and protein modifi-
cation and stability. The most immediate and well-stud-
Gene expression is a key determinant of phenotype. The ied level of control of gene expression is transcription
sequences and factors controlling when and how much initiation. Molecular technology has led to an extensive
protein is synthesized are equally as important as the study of transcription initiation, so a large amount of
DNA sequences encoding the amino acid makeup of information on gene expression refers to this level of
a protein. Early studies aimed at the characterization transcription.
of gene structure were confounded by phenotypes that
resulted from aberrations in gene expression rather than Regulation of Messenger RNA Synthesis
in protein structural alterations. at Initiation
Two types of factors are responsible for regulation of
mRNA synthesis: cis factors and trans factors (transcrip-
Advanced Concepts tion factors; Fig. 2.2). Cis factors are DNA sequences
that mark places on the DNA involved in the initiation
With advances in crystallography, the molecu- and control of RNA synthesis. Trans factors are proteins
lar mechanisms of RNA synthesis were revealed. that bind to the cis sequences and direct the assembly
During transcription, DNA enters a positively of transcription complexes at the proper gene. In order
charged cleft between the two largest subunits of for transcription to occur, several proteins must assem-
the RNA polymerase. At the floor of the cleft is ble at the gene’s transcription initiation site, including
the active site of the enzyme to which nucleotides specific and general transcription factors and the RNA
polymerase complex.
5′
Cis element
3′ Histooricaal Higghlligghtts
3′ 5′
DNA The production of particular proteins by bacte-
Trans
factor ria growing in media containing specific sub-
strates was observed early in the last century, a
phenomenon termed enzyme adaptation, later
called induction. Detailed analysis of the lactose
operon in E. coli was the first description of an
5′ 3′ inducible gene expression at the molecular level.
3′ 5′ The effect of gene expression on phenotype was
FIGURE 2.2 Cis elements are DNA sequences that are recog- initially demonstrated by Monod and Cozen-
nized by regulatory proteins (trans factors). Binding of trans Bizare in 1953 when they showed that synthesis of
factors can turn gene expression on or off. tryptophan in Aerobacter was inhibited by trypto-
phan.9 Jacob and Monod subsequently introduced
the concept of two types of genes, structural and
regulatory, in the lactose operon in E. coli.10
An operon is a series of structural genes transcribed
together on one mRNA and subsequently separated into
individual proteins. In organisms with small genomes, Figure 2.3 shows a map of the lac operon. The three
such as bacteria and viruses, operons bring about the structural genes of the operon are copied into a single
coordinated expression of proteins required at the same transcript under the control of the operator.
time, for example, the enzymes of a metabolic pathway. The sequences coding for the repressor protein are
For example, the lactose operon (lac operon) in Esch- located just 5′ to the operon. In the absence of lactose,
erichia coli contains three structural genes: lacZ, lacY, the repressor protein binds to the operator sequence and
and lacA, which are all required for the metabolism of prevents transcription of the operon (Fig. 2.4A). When
lactose. The lacZ gene product, β-galactosidase, hydro- lactose is present, it binds to the repressor protein and
lyzes lactose into glucose and galactose. The lacY gene changes its conformation and lowers its affinity to bind
product, lactose permease, transports lactose into the the operator sequence. This results in expression of the
cell. The lacA gene product, thiogalactoside transacety- operon (Fig. 2.4B). Jacob and Monod originally deduced
lase, transacetylates galactosides. A lacI gene encodes these details through analysis of a series of mutants
a protein repressor that binds to the lacO cis factor in (changes in the DNA coding for the various components
the DNA (the operator) just 5′ to the start of the operon of the operon).11 Since their work, numerous regulatory
near where RNA polymerase binds (lacP). When E. coli systems have been described in prokaryotes and eukary-
is growing on glucose as a carbon source, the lactose- otes, all using the same basic idea of combinations of cis
metabolizing enzymes are not required, and this operon and trans factors.
is minimally expressed. Within two to three minutes after Other operons are controlled in a similar manner by
shifting to a lactose-containing medium, the expression the binding of regulatory trans factors to cis sequences
of these enzymes is increased a thousand-fold. preceding the structural genes (Fig. 2.5A). A different
Regulator gene P O lacZ lacY lacA

5′ 3′
3′ 5′
DNA
FIGURE 2.3 General structure of the lac operon. The regulator or repressor gene codes for the repressor protein trans factor that
binds to the operator.
Repressor RNA
polymerase
5′ 3′
3′ 5′
DNA Regulator P O lacZ lacY lacA
A gene
Inducer
(lactose)
5′ 3′
3′ 5′
DNA Regulator P O lacZ lacY lacA
gene
B
FIGURE 2.4 Two states of the lac operon. (A) The repressor protein (R) binds to the operator cis element (O) preventing tran-
scription of the operon from the promoter (P). (B) In the presence of the inducer lactose, the inducer binds to the repressor, chang-
ing its conformation and decreasing its affinity for the operator, allowing transcription to occur.
Inducer
Repressor
5′ 3′
3′ 5′
P O
A
Corepressor
Repressor
5′ 3′
3′ 5′
P O
B
Activator
5′ 3′
3′ 5′
P O
C
FIGURE 2.5 Modes of regulation in prokaryotes include induction as found in the lac operon (A), repression as found in the arg
operon (B), and activation as in the mal operon (C).
type of negative control is that found in the arg operon,

where a corepressor must bind to a repressor in order to Advanced Concepts
turn off transcription (enzyme repression; Fig. 2.5B).
Compare this with the inducer that prevents the repres- Unlike prokaryotes, eukaryotes do not have
sor from binding the operator to turn on expression of operons. Coordinately expressed genes can be
the lac operon (enzyme induction). The mal operon scattered in several locations. Synchronous expres-
is an example of positive control where an activator sion is brought about in eukaryotes by combinato-
binds with RNA polymerase to turn on transcription rial control; that is, genes that are expressed in a
(Fig. 2.5C). similar pattern share similar cis elements so that
Another mechanism of control in bacteria is atten- they respond simultaneously to specific combina-
uation. This type of regulation works through the for- tions of controlling trans factors.
mation of stems and loops in the RNA transcript by
intrastrand hydrogen bonding of complementary bases
(Fig. 2.6). These secondary structures allow or prevent Antitermination Termination
transcription, for instance, by respectively exposing or
sequestering ribosome-binding sites at the beginning of
the transcript. 1
The general arrangement of cis factors in prokaryotes 2 3 1
and eukaryotes is shown in Figure 2.7. These sequences
4
are usually 4 to 20 base pairs (bp) in length. Some are Trp Trp Trp
3 4
inverted repeats with the capacity to form a cruciform Trp Trp Trp
structure in the DNA duplex recognizable by specific
proteins. Prokaryotic regulatory sequences are usually
FIGURE 2.6 Attenuation regulates expression at the level of
found within close proximity of the gene. Eukaryotic translation. In addition to repression of RNA synthesis,
genes have both proximal and distal regulatory elements. enzymes encoded in the trp operon are also regulated at the
Distal eukaryotic elements can be located thousands of level of translation. With low tryptophan, the ribosome pauses
base pairs away from the genes they control. Enhancers at trp codons in the mRNA, allowing the anti-terminator
and silencers are examples of distal regulatory elements hairpin to form by hybridization of RNA regions 2:3, and
that, respectively, stimulate or dampen the expression of translation continues. When tryptophan levels are adequate,
distant genes (Fig. 2.8). the ribosome moves quickly through the trp codons, inducing
hybridization of RNA regions 3:4 and termination of
translation.
Prokaryotes
Proximal elements Structural gene
5′ 3′
3′ 5′
Promoter
Eukaryotes
Distal elements Proximal elements Structural gene
5′ 3′
3′ 5′
Promoter
FIGURE 2.7 Cis regulatory elements in prokaryotes are located close to the structural genes they control in the vicinity of the
promoter. In eukaryotes, distal elements can be located thousands of base pairs away from the genes they control. Proximal ele-
ments can be located in or around the genes they control. Elements may also be located behind their target genes.
Coactivator sequence structure and the presence of exonucleases

(histone acetyl and endonucleases that digest the RNA. Enzymatic and
transferase)
structural alterations in RNA interfere with its proc-
essing in eukaryotes and its translation into protein.12
In eukaryotes, RNA processing steps will affect the
Activator
production of protein and ultimately the phenotype.
Alternate splicing in combination with protein factors is
DNA responsible for many tissue- and development-specific
Regulatory region gene-expression patterns (e.g., during hematopoiesis).13
Nucleosome
TFIID
Regulatory region (TATA-binding
protein)
Advanced Concepts
Circular RNA structures (circular RNA) are impli-
Enhancer Promoter cated in the control of gene expression, among a
5′ 3′ variety of other cellular functions.14,15 These RNAs,
3′ 5′
DNA Regulator first thought to be viral products, are endogenous
Activator protein
protein by-products of RNA splicing in protein-coding
Other basal genes. Their roles include protein binding, activa-
factors
TFIID tion of transcription, and subcellular localization.
5′ 3′ They are naturally stable because they do not have
3′ 5′ ends subject to exonucleases. They are implicated
RNA
polymerase II in tissue development, stress response, cancer, and
other diseases.13,16
TFIID
5′ 3′ In prokaryotes, RNA transcription and translation are
3′ 5′
concurrent. This protects the RNA from the processing
and exogenous factors. Just as in eukaryotes, however,
RNA stability in prokaryotes is affected by secondary
structure (folding of the RNA molecule) and polyade-
nylation of the 3′ end of the transcripts.17 Codon usage
TFIID
and cofactor availability may also alter the speed of
3′
5′ translation, resulting in decreased or increased amounts
of protein.18
FIGURE 2.8 Interaction of transcription factors and histone
acetylation at the regulatory region of a gene (top) induce Post-Translational Regulation
assembly of protein factors and RNA polymerase at the pro-
moter (bottom). Once proteins are synthesized, the genes might be consid-
ered successfully expressed; however, post-translational
events influence the function of the completed protein.
Some nascent peptides and proteins are modified with
Post-Transcriptional Regulation
lipids, sugars, and other factors by cellular enzymes in
For most genes, the RNA transcript has to be translated order to function. Protein-modifying enzymes include
to protein to bring about phenotype. The transcript must phosphatases, kinases, ubiquitinases, transferases, acety-
be processed and translated into protein. Several factors lases, methylases, and ligases. As proteins move through
affect the stability of the RNA transcript, including RNA the endoplasmic reticulum and Golgi, they are directed
to their functional locations or marked for secretion. By modification, nucleic acid methylation, and noncoding
controlling protein function, these activities are import- RNA.
ant for cell phenotype and behavior. Signals in Golgi
have even been implicated in oncogenic transformation.19
Histone Modification
Gene expression is measured in the laboratory at the
level of transcription and protein production. Single or Chromatin is nuclear DNA and its associated pro-
multiple genes are tested, depending on the nature of the teins. In eukaryotes, the double helix of chromosomal
test or study. The range of activities and interactions that DNA is compacted onto nucleosomes. A nucleosome is
participate in the production and function of RNA and about 150 bases of DNA wrapped around a complex of
proteins complicate the interpretation of gene expres- eight histone proteins, two each of histone 2A, histone
sion and its control. In addition to expression levels, 2B, histone 3, and histone 4. Histones are not only struc-
changes in the DNA sequence (mutations) can result in tural proteins but also regulate access of trans factors and
successfully expressed and modified transcripts or pro- RNA polymerase to the DNA helix (Fig. 2.9). Modifica-
teins without function. This and other possibilities must tion of histone proteins affects the activity of chromatin-
be taken into account when interpreting gene-expression associated proteins and transcription factors that increase
results. or decrease gene expression. Through these protein inter-
actions, chromatin can move between transcriptionally
active and transcriptionally silent states. Different types
EPIGENETICS of histone modifications, including methylation, phos-
phorylation, ubiquitination, and acetylation, establish a
In 1942, Conrad Waddington, an embryologist, defined complex system of control. Modifications have specific
epigenetics as a developmental phenomenon that effects on chromatin; for example, acetylation lowers
allowed cells to take on different phenotypes (differ- the positive charge of the histones, decreasing binding
entiate) without changes in the genetic structure. Epi- strength to the negatively charged DNA, thereby making
genetics was a way to explain how a single cell could the DNA more available for interaction with transcription
differentiate into a multicellular organism without vast factors and RNA polymerase (open chromatin). Methyl-
genotypic changes. He related this idea to the notion of ation of histones attracts enzymes that further methylate
“epigenesis,” the 17th-century concept of the complex DNA, resulting in decreased gene expression (closed
interactions between Mendel’s genotype and pheno- chromatin). Phosphorylation, ubiquitination, and other
type.20 Sixteen years later, microbiologist David Nanney modifications directly affect the histone–DNA interaction
proposed an alternate two-part system of cellular control, or recruit other modifying enzymes (Table 2.3).
one based on genetics (DNA) and the other determining Modifications occur on specific amino acids, and
which genes would be expressed at which time.21 In this carboxy ends the histone protein sequences (Fig. 2.10).
model the term epigenetics was used to emphasize the These “histone tails” extend from the nucleosome, where
reliance of the auxiliary system on the genetic informa- they are available to the acetylases, methylases, and other
tion. With the subsequent growth in understanding of the enzymes. The histone modifications (histone marks)
nature of the regulation of gene expression, epigenetics form an environment for interaction with other chroma-
was further defined as mitotically and meiotically her- tin-binding factors and, ultimately, regulatory proteins.22
itable changes in phenotype not encoded in genotype. A “histone code” has been proposed, analogous to the
Epigenetics may be a mechanism of rapid and heritable genetic code, correlating specific histone modifications
adaptation to environmental changes without alteration with biological effects (Table 2.1).23,24 The recommended
in the DNA genotype. The current study of epigenetics nomenclature for core histone (H2A, H2B, H3, H4)
involves chemical and structural modifications in chro- modifications is the name of the histone followed by the
matin and the activity of particular noncoding RNAs. amino acid and its position in the protein using the single-
Changes in gene expression and phenotype as a result of letter code, followed by the modification. Acetylation
aberrant epigenetic phenomena are studied in a variety of lysine at position 27 of histone H3 would be desig-
of disease states. These phenomena include histone nated H3K27Ac. Methylation of lysine at position 20
Promoter
Activator
proteins
pol II
FIGURE 2.9 Histones are components of nucleosomes required for organization of DNA in the nucleus. They are also important
regulators of gene expression. In order for trans factors, such as activators, RNA polymerase, and its cofactors, to access DNA,
histones must release the cis sequences and promoter to which they bind.
of histone H2B would be H2BK20Me.The involvement of more than 200 bp in length with an observed/expected
of histone modification with gene expression has led to ratio of the occurrence of CpG of greater than 0.6.26
the study of aberrations in these modifications in disease This definition may be modified to a more selective GC
states such as viral infections and neoplastic cells. Ther- content to exclude unrelated regions of naturally high
apeutic agents such as histone deacetylase inhibitors are GC content.27 CpG islands are found around the first
currently in use for some hematological malignancies.25 exons, promoter regions, and sometimes toward the
Thus, histone modifications may be another target for 3′ ends of genes. There are over 45,000 CpG islands in
diagnostic, prognostic, and therapeutic applications. the human genome. Aberrant DNA methylation at these
sites is a source of dysregulation of genes in disease
states. Methylation of cytosine residues in the promoter
Nucleic Acid Methylation regions of tumor-suppressor genes is a mechanism of
inactivation of these genes in cancer.28
DNA Methylation
Methylation of DNA is the main mechanism of
DNA methylation is another type of epigenetic regula- genomic imprinting, the stage- and gamete-specific
tion of gene expression in eukaryotes and prokaryotes. silencing of genes.29 Imprinting maintains the balanced
In vertebrates, methylation of DNA occurs in cytosine- expression of genes in growth and embryonic develop-
guanine–rich sequences in the DNA (CpG islands; ment by selective methylation of homologous genes. This
Fig. 2.11). CpG islands were initially defined as regions controlled methylation occurs during gametogenesis and
There are four enzymes that methylate DNA in

TABLE 2.3 The Histone Code mammals: DNA methyltransferase 1 (DNMT1), DNA
methyltransferase 3a/3b (DNMT3), and DNA meth-
Histone Modification Function yltransferase 3L (DNMT3L). A fifth enzyme, DNA
H2A Acetylation Activation methyltransferase 2, may be involved in modifying bases
in tRNA or other targets. DNMT1 is a maintenance
H2A Phosphorylation Mitosis, DNA repair* enzyme and mainly methylates hemimethylated DNA,
H2A Ubiquitylation Stem cells
that is, cytosines that are methylated on one strand of
the double helix and not the other. This is the state of
H2B Acetylation Activation methylated DNA after replication where the newly syn-
thesized strand is not methylated like the parent strand.
H2B Phosphorylation Apoptosis
DNMT3 is a de novo methylase and methylates un-
H2B Ubiquitylation Transcription methylated DNA, establishing newly methylated regions,
as in imprinting. DNMT3L is likely nonfunctional as a
H3 Unmodified Silencing
methylase because it lacks a functional catalytic site. It
H3 Methylation Silencing/activation* may regulate DNMT3A and 3B activities by occupying
DNMT3A and 3B protein or DNA-binding sites.
H3 Phosphorylation Mitosis/activation*
DNMT1 and DNMT3 recognize cytosine bases in
H3 Acetylation Activation/histone DNA followed by guanine bases (CG or CpG). The
positioning* symmetrical nature of the CG sequence reflects the sym-
H4 Phosphorylation Activation
metry of the enzymes. The complement of 5′-CG-3′ is
5′-CG-3′. The enzymes are recruited to potentially meth-
H4 Methylation Silencing/activation* ylated areas by proteins that bind modified histones as
well as proteins that bind hemimethylated DNA.
H4 Acetylation DNA repair/histone
positioning
*Function depends on which amino acid is modified or if a combination of Advanced Concepts

modifications is present.
One theory holds that a preexisting and gene-

is different in male and female gametes. A convenient specific histone code extends the information
illustration of imprinting is the comparison of mules and potential of the genetic code.24 Accordingly,
hinnies. A mule (progeny of a female horse and male euchromatin, which is transcriptionally active,
donkey) has a distinct phenotype from that of a hinny has more acetylated histones and less methylated
(progeny of a male horse and a female donkey). The dif- histones than transcriptionally silent heterochro-
ference is due, in part, to the distinct imprinting of genes matin made up of more condensed nucleosome
inherited through egg formation (oogenesis) versus fibers. Although methylated histones can activate
those inherited through sperm formation (spermatogene- transcription by recruiting histone acetylases, the
sis). Genetic diseases in humans, such as Angelman syn- establishment of localized areas of histone meth-
drome and Prader–Willi syndrome, are clinically distinct ylation can also prevent transcription by recruiting
conditions that result from the same genetic defect on proteins for DNA methylation and heterochroma-
chromosome 15.30 The phenotypic differences depend tin formation. This is one form of gene or tran-
on whether the genetic lesion involves the maternally scriptional silencing.22 Transcriptional silencing is
or paternally inherited chromosome. Imprinting may be responsible for inactivation of the human X chro-
partly responsible for the abnormal development and mosome in female embryo development and posi-
phenotypic characteristics of cloned animals because the tion effects, the silencing of genes when placed in
process of cloning by nuclear transfer bypasses egg and heterochromatic areas.
sperm formation (gametogenesis).31,32
H2N-ARTKQTARKSTGGKAPRKQLATKAARKSAP H3
2 4 9 10 14 17 18 23 26–28
H2N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT H4
1 3 5 8 12 16 20
H2N-MSGRGKGGGKAR QAKTVAKKSGKKPAPASKAPEPM-NH2
2 6 10 21 16 13 6
H2A H2B
HO2C-KSKAKHS VTKYTSSK-CO2H
120 121
Ac HAT
HDAC Me Ac Acetylation
Me
OP Ac OP
Ac
Deacetylation,
methylation Me Ac Ac
Me
FIGURE 2.10 Histones H2A, H2B, H3, and H4 are modified at specific amino acids on the amino terminal and carboxyterminal
ends (top). Depending on the modifications (histone marks), the associated DNA will form closed, nonexpressed heterochromatin
(left) or open, actively transcribed euchromatin (right).
…GGAGGAGCGCGCGGCGGCGGCCAGAGA
the vast intergenic regions of DNA and in gene
bodies (coding sequences), methylation is much
AAAGCCGCAGCGGCGCGCGCGCACCCGGA heavier. Abnormal hypermethylation of CpG
islands as well as hypomethylation of intergenic
areas can occur. In some types of colon and brain
CAGCCGGCGGAGGCGGG…
cancer, hypermethylation of multiple CpG islands
FIGURE 2.11 CpG islands are sequences of DNA rich in the has been defined as a CpG Island Methylator Phe-
C-G dinucleotides. These structures have no specific sequence notype (CIMP) with diagnostic and prognostic
other than a higher-than-expected occurrence of CpG. implications.33 Hypomethylation in the much larger
intergenic areas results in an overall hypomethyl-
ation in tumor cells. Hypomethylation around ret-
Advanced Concepts rotansposons such as the long interspersed nuclear
elements (LINEs) is thought to be a cause of
Normally, DNA is unmethylated or transiently the genomic instability (chromosome alterations,
methylated in CpG islands before active genes. In breakage, and loss) seen in some cancers.
DNA Demethylation but not in the 3′ polyA tails. Methyltransferase enzymes

encoded by METTL14 and METTL3 genes recognize
In normal cells, DNA methylation, especially in CpG
and methylate target RNA regions.37 RNA methylation
islands, is reversible. In this way, DNA methylation can
affects RNA stability, splicing, and translation.
serve as an additional layer of transcriptional control
in addition to cis and trans genetic factors. DNA meth-
ylation occurs passively through rounds of DNA rep-
lication, enzymatic alteration, and repair or by active
CLASSIFICATION OF EPIGENETIC FACTORS
conversion.34 The major pathway of active demethyl-
Histone and nucleic acid modifications that bring about
ation in vertebrate cells is a series of oxidative reac-
epigenetic processes have been classified as writers and
tions catalyzed by the Ten Eleven Translocation (TET)
erasers. Methyltransferases, phosphorylases, acetylases,
enzymes. These enzymes first convert 5-methylcytosine
and other enzymes that modify histones, RNA, and
to 5-hydroxymethylcytosine, which is then converted to
DNA are the writers. Deacetylases, TET enzymes, and
5-formylmethyl cytosine and then to 5-carboxymethyl
enzymes that reverse these modifications are classified
cytosine. A decarboxylase enzyme catalyzes the final
as erasers. A third category of readers is the transcription
step to restore 5-methylcytosine.
factors, methyl-group–binding proteins and others that
recognize the state of chromatin and act accordingly.
Advanced Concepts Another functional classification for epigenetics in
cancer cells has also been proposed. This classifica-
The process of demethylation catalyzed by the TET
tion considers the interrelationship between genetic and
enzymes connects the epigenetic state of DNA with
epigenetic factors. Classes include modulators, modifi-
metabolic processes.35 The TET enzymes require
ers, and mediators. Modulators are those activities that
alpha-ketoglutarate in order to function. Alpha-
activate the epigenetic process. This category includes
ketoglutarate is a product of the isocitrate dehy-
genetic factors such as IDH1/2 and the KRAS and TP53
drogenase enzymes (IDH1 and IDH2), which are
cancer genes. Modifiers are the writers and erasers of the
part of the citric acid cycle. Specific amino acid
previous system that change or maintain the chromatin
substitution mutations in the IDH enzymes replace
structure. Modulators are the readers, such as transcrip-
the normally produced a-kg with 2-hydroxy
tion factors, that ultimately bring about the effects of the
glucose. The 2-hg competes with a-kg, causing
chromatin state.
loss of function of the TET enzymes. Thus, com-
The latter system emphasizes the concept that epi-
ponents of the metabolic citric acid cycle can alter
genetics is not completely independent of genetic control
the methylation state of DNA. DNA alterations in
factors and vice versa. The combination of genetic and
the IDH enzymes are found in cancer (e.g., glio-
epigenetic activities forms a complex system that con-
blastoma and acute myeloid leukemia).
trols gene expression and ultimately phenotype through
development, growth, and survival responding to envi-
ronmental influences.
RNA Methylation
There are more than 100 post-transcriptional modifica-
tions on RNA.36 Transfer RNA has a number of altered NONCODING RNAs
and methylated bases, increasing the recognition of
charging enzymes and codon–anticodon binding. Mes- Noncoding RNAs carry out their function as RNA and
senger RNA, ribosomal RNA, and noncoding RNA are are transcribed but not translated into protein. Noncod-
also methylated. The methylated bases are most fre- ing RNAs were thought to be “leaky” transcription from
quently N6-methyladenosine (m6A) in a three-base rec- intergenic areas of the genome. These species of RNA
ognition site, GAC, and less commonly AAC. About are now known to have roles in gene regulation and
25% of mRNAs have m6A clustered around stop codons, even in information transfer across generations.38 Non-
sometimes found in 5′ untranslated regions of the RNA coding RNAs are found in oocytes and spermatozoa.
Piwi RNA (piRNA), which affects transposon transcrip- eukaryotic cells against viral invasion. In a process that
tion in germ cells, is specific to gamete-mediated epi- is not yet completely understood, double-stranded RNA
genetic inheritance.39 (dsRNA) species are believed to originate from tran-
Noncoding RNAs are classified as small, interme- scription of inverted repeats or by the activity of cellular
diate, and long, depending on their length. Small non- or viral RNA–directed RNA polymerases. Biochemical
coding RNAs, microRNAs (miRNAs), short interfering analysis of RNA interference has revealed that these
RNAs (siRNAs), and long noncoding RNAs (lncRNAs) 21- to 22-nucleotide dsRNAs are derived from succes-
are regulators of gene expression. sive cleavage of dsRNAs that are 500 or more nucleo-
tide base pairs in length.48 The ribonuclease III enzyme,
dicer, is responsible for the generation of siRNA and
MicroRNAs
another small RNA, microRNA (see following discus-
MicroRNAs (miRNAs) are regulatory RNAs, 17 to sion), from dsRNA precursors.49 siRNAs are not nor-
27 nucleotides (nt) in length, derived from endoge- mally found in animal cells. They are frequently used
nous RNA hairpin structures (RNA folded into double- in the laboratory to artificially turn off the expression
stranded states through intrastrand hydrogen bonds). of specific genes. Research using siRNAs is a standard
MicroRNAs were discovered in the worm Caenorhabdi- approach to exploring the function and activity of genes
tis elegans. Two 22-nt RNAs were shown to contribute of interest.
to the temporal progression of cell fates by triggering
down-regulation of target mRNAs.40–42 These RNA
Other Small RNAs
species were also called small temporal RNAs (stRNAs).
Functionally, miRNAs control gene expression by Since the late 1990s, increasing varieties of small
pairing with imperfectly complementary sequences in RNAs (sRNAs) have been described in prokaryotes and
mRNAs to inhibit translation. Many miRNAs have been eukaryotes, including tiny noncoding RNAs (tncRNAs,
identified in eukaryotic cells and viruses.43–45 Bacteria 20 to 22 b), small modulatory RNAs (smRNAs, 21 to
have genes that resemble miRNA precursors; however, 23 b), small nucleolar RNAs (snoRNAs), tmRNAs,
the full miRNA system has not been demonstrated in guide RNAs (gRNAs), and others. In addition to RNA
bacteria. Over 5,600 miRNAs have been identified in synthesis and processing, these molecules influence
humans.46 numerous cellular processes, including plasmid replica-
MicroRNAs perform diverse functions in eukaryotic tion, bacteriophage development, chromosome structure,
cells, affecting gene expression, cell development, and and development. These small, untranslated RNA mol-
defense. Because production of miRNAs is strictly reg- ecules have been termed sRNAs in bacteria and noncod-
ulated as to time or stage of cell development, finding ing RNAs (ncRNAs) in eukaryotes.
them is a technical challenge. Many of these species are
only present in virally infected cells or after the intro-
duction of foreign nucleic acid by transformation. Novel Advanced Concepts
approaches are being applied to the discovery of rare
miRNAs expressed in specific cell types at specific times. The first demonstration of directed RNA inter-
miRNA dysregulation is associated with a variety of ference in the laboratory occurred in the experi-
diseases, especially cancer, where miRNAs promote the ments of Fire et al. with Caenorhabditis elegans.50
tumor-cell phenotype (oncomirs). Molecular mimics of These investigators injected dsRNA into worms
miRNA and molecules targeted at miRNAs (antimirs) and observed dramatic inhibition of the genes
are being tested as therapeutic agents.47 that generated the RNA. Since then, siRNAs have
been introduced into plants and animals, including
human cells growing in culture. Injection of long
Small Interfering RNAs
dsRNA can kill human cells, but gene silencing
Small interfering RNAs (siRNAs) are the functional can be achieved by the introduction of siRNAs
intermediates of RNA interference (RNAi), a defense in
or plasmids coding for their dsRNA. Librar- Advanced Concepts

ies of siRNAs or DNA plasmids encoding them
have been made that are complementary to over RNA interference has been utilized as a method to
8,000 human genes.51 These genetic tools have specifically inactivate genes in the research labo-
potential applications not only in identifying genes ratory. Preferable to gene deletions or “knock-out”
involved in disease but also in the treatment of methods that take months to inactivate a single
some of these diseases, particularly cancers where gene, RNAi can specifically inactivate several
overexpression or abnormal expression of specific genes in a few hours.
genes is part of the tumor phenotype. Initially, this method did not work well in mam-
malian cells, due to a cellular response elicited by
the introduction of long dsRNAs that turns off
Regulation of Gene Expression by MicroRNAs multiple genes and promotes cell suicide. Adjust-
ments to methods, such as using shorter dsRNAs,
Thousands of microRNAs encoded among genes have been demonstrated to work in mammalian
in higher eukaryotes regulate gene expression post- cell cultures, but the efficiency of silencing will
transcriptionally by binding to the 3′ end of mRNA vary among cell lines and different experimental
and preventing its translation into protein (Fig. 2.12A). conditions.55 This type of specific gene control
MicroRNAs are transcribed as larger precursors (pre- in vitro may lead to directed control of transcrip-
miRNAs), which are cleaved into hairpins by RNase tional states, which would be useful in the clinical
III–like endonucleases, dicer and Drosha. The hairpins laboratory setting as well as the research labora-
are further digested into short, single-stranded RNA tory. Because of the high specificity of siRNAs,
imperfectly complementary to the 3′ end of the target RNAi has also been proposed as a manner of gene
gene.52 Hybridization between the microRNA and the and viral therapy.56 Silencing has been targeted to
mRNA prevents translation of the target RNA and even- growth-activating genes such as the vascular endo-
tually results in degradation of the mRNA. thelial growth factor in tumor cells. Small interfer-
ing RNA silencing may also be aimed at the HIV
Regulation of Gene Expression and influenza viruses. As with other gene-therapy
by RNA Interference methods, the stability and delivery of the therapeu-
Another phenomenon that affects transcription is RNA tic siRNAs are major challenges.57
interference, or RNAi. First discovered in the worm
C. elegans in 1993, RNAi is not present in humans;
however, it is a useful tool in the research laboratory
Long Noncoding RNAs
to selectively eliminate expression of specific genes
in vitro. RNAi is mediated through siRNAs.53 Like Long noncoding RNAs (lncRNAs) are defined by their
microRNA, the siRNAs and other proteins assem- length of 200 to 100,000 bases. lncRNAs are impor-
ble into an enzyme complex, called the RNA-induced tant regulators of chromatin structure, affecting gene
silencing complex (RISC; see Fig. 2.12B). The RISC expression and disease processes. Genomic analysis has
uses the associated siRNA to bind and degrade mRNA revealed from 15,000 to as many as 58,648 lncRNAs
with sequences exactly complementary to the siRNA. encoded in the human genome.58,59
Translation of specific genes can thus be inhibited. lncRNAs function in several ways (Fig. 2.13).60
Alternatively, siRNAs may bind to specific sequences Through secondary structure (folding into dou-
in already-transcribed homologous RNA, targeting them ble-stranded domains), lncRNAs can bind proteins that
for degradation into more siRNAs. Although the reg- normally would bind to cis sequences in DNA, prevent-
ulatory mechanisms are similar, regulation by siRNA ing their regulatory action. Multiple domains or hairpins
requires the introduction of foreign RNA into the cell, in the lncRNAs can act as a scaffold to bring proteins
whereas microRNAs are encoded in the cell’s DNA.54 together. Double- and single-stranded domains can bind
Trigger dsRNA
Dicer
siRNA
pol II
RISC
5′ cap 7-methylG 5'ppp 5' G/A AAAAAA
pri-miRNA
Target mRNA
RNase III
(Drosha)
RISC
RNase III
(Dicer)
Direct cleavage
RISC
microRNA
A B Target mRNA
FIGURE 2.12 (A) In miRNA silencing, miRNA transcripts are transcribed from host genes, processed, and joined to the RNA-
Induced Silencing Complex (RISC) assembly. (B) In siRNA silencing, a trigger double-stranded RNA is cleaved into siRNAs that
become part of the RISC assembly. Led by the complementary siRNA sequences, RISC binds to the target RNA and begins RNA
cleavage.
proteins to specific DNA-binding sites. Enhancers are

cis sequences found far away from the genes that they Histooricaal Higghlligghtts
control. Through scaffolding, lncRNAs may connect
trans factors bound to the enhancer region to trans In 1904, Austrian zoologist Paul Kammerer
factors bound to proximal cis sites. lncRNAs may also wanted to show that animals acquire new charac-
bind small ligands, interact with miRNA in RISC com- teristics when exposed to different environments
plexes, form triplex structures with double-stranded and that these changes are heritable. He published
DNA, or be further processed into small RNA fragments many papers and books describing environmen-
through RNAseP cleavage at the lncRNA 3′ ends. tally induced phenotypic changes passed down
ncRNAs are associated with disease states and are through multiple generations in amphibians and
potential therapeutic targets.61 The study of the biolog- reptiles. These observations might have provided
ical roles of both small and long ncRNAs has led to the first reports of epigenetic inheritance. Kam-
efforts in the use of ncRNAs as therapeutic targets. The merer ’s work was controversial, however, with
continued clarification of ncRNA mechanisms will lead accusations of photographic image manipulation.
to effective ncRNA drug design. Furthermore, there was limited success of other
Decoy Scaffold
Guide Enhancer
Gene
Adapter
FIGURE 2.13 lncRNAs can titrate away DNA-binding proteins from cis sites or act as scaffolds to bring proteins into a complex.
They may recruit transcription factors or histone-modification enzymes through RNA–DNA interactions or RNA interactions with
DNA-binding proteins. lncRNAs may connect enhancer sites to proximal sites through protein–RNA and protein–DNA interac-
tions. (Reprinted with permission, Rinn JL, Chang HY. Genome regulation by long noncoding RNAs. Annual Review of Biochem-
istry 2012;81[1]:145–66.)
researchers in replicating Kammerer ’s findings

on environmentally induced heritable foot-pad complex that initiates methylation of the chromo-
changes in toads and color changes in salaman- some. This mechanism silences nearly all genes
ders.62 With the currently accepted molecular on one X chromosome. Another lncRNA, Tsix, is
basis for epigenetics, researchers are now revisit- complementary to Xist and extends across the Xist
ing Kammerer ’s early ideas.63 locus. Tsix negatively regulates Xist as an anti-
sense RNA.65
Advanced Concepts
A special lncRNA, Xist (X-inactive specific tran- STUDY QUESTIONS
script) is important for X-chromosome inactiva-
tion in mammalian female (XX) cells.64 Along
with other ncRNA and proteins, it is expressed Gene Expression
from a region of the X chromosome, the X inac-
tivation center (XIC). These products form a 1. Proteins that bind to DNA to control gene
expression are called __________ factors.
2. The binding sites on DNA for proteins that control 2. How does the complementarity of siRNA to its
gene expression are _________ factors. target mRNA differ from that of miRNA?
3. How might a single mRNA produce more than one 3. What is imprinting in DNA?
protein product?
4. What sequence structures in DNA, usually found
4. The type of transcription producing RNA that 5′ to structural genes, are frequent sites of DNA
is continually required and relatively abundant methylation?
in the cell is called ______________________
transcription. 5. What is the RISC?
5. A set of structural genes transcribed together on one 6. Name four functions of lncRNAs.
polycistronic mRNA is called a(n) _________.
The Lac Operon References

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Section II
Common Techniques
in Molecular Biology
77
Chapter 3
Nucleic Acid Extraction Methods
Outline MEASUREMENT OF NUCLEIC ACID QUALITY AND QUANTITY

Electrophoresis
ISOLATION OF DNA Spectrophotometry
Preparing the Sample Fluorometry
Bacteria and Fungi Microfluidics
Viruses
Nucleated Cells in Suspension (Blood and Bone Marrow
Aspirates)
Plasma
Tissue Samples
DNA Isolation Chemistries Objectives
Organic Isolation Methods
Inorganic Isolation Methods 3.1 Compare and contrast organic, inorganic, and
Solid-Phase Isolation solid-phase approaches for isolating cellular and
Proteolytic Lysis of Fixed Material mitochondrial DNA.
Rapid Extraction Methods 3.2 Describe DNA isolation from minimal and
Isolation of Mitochondrial DNA challenging samples.
ISOLATION OF RNA 3.3 Compare and contrast organic and solid-phase
Total RNA approaches for isolating total RNA.
Specimen Collection 3.4 Distinguish between the isolation of total RNA and
RNA Isolation Chemistries that of messenger RNA.
Organic Isolation 3.5 Describe the gel-based, spectrophotometric, and
Solid-Phase Isolation fluorometric methods used to determine the
Proteolytic Lysis of Fixed Material quantity and quality of DNA and RNA preparations.
Isolation of polyA (Messenger) RNA 3.6 Calculate the concentration and yield of DNA and
RNA from a given nucleic acid preparation.
78
Chapter 3 • Nucleic Acid Extraction Methods 79
The purpose of nucleic acid extraction is to release the

nucleic acid from the cell for use in subsequent proce- TABLE 3.1 Yield of DNA From Different
dures. Ideally, the target nucleic acid should be free of Specimen Sources29-35
contamination with protein, carbohydrate, lipids, or other
nucleic acids, that is, DNA free of RNA or RNA free Expected
Specimen Yield*
of DNA. The initial release of the cellular material is
achieved by breaking the cell wall (if present) and cell Specimens Adequate for Analysis Without DNA Amplification
and nuclear membranes (cell lysis). Lysis must take place
in conditions that will not damage the nucleic acid. Fol- Blood† (1 mL, 3.5–10 × 106 WBCs/mL) Buffy 50–200 μg
coat† (1 mL whole blood)
lowing lysis, the target material is purified, and then the
concentration and purity of the sample can be determined. Bone marrow† (1 mL) 100–500 μg
Cultured cells (107 cells) 30–70 μg
ISOLATION OF DNA Solid tissue‡ (1 mg) 1–10 μg
Lavage fluids (10 mL) 2–250 μg

Although Miescher first isolated DNA from human cells
in 1869 by precipitation,1 the early routine laboratory Mitochondria (10-mg tissue, 10 cells) 7
1–10 μg
procedures for DNA isolation were developed from
density-gradient centrifugation strategies. Meselson and Plasmid DNA, bacterial culture (100-mL 350 μg–1 mg
overnight culture)
Stahl used such a method in 1958 to demonstrate semi-
conservative replication of DNA.2 Later procedures took Bacterial culture (0.5 mL, 0.7 absorbance 10–35 μg
advantage of solubility differences among chromosomal units)
DNA, plasmids, and proteins in alkaline buffers. Large Feces§ (1 mg; bacteria, fungi) 2–228 μg
(greater than 50 kbp) chromosomal DNA and proteins
cannot renature properly when neutralized in acetate at Specimens Adequate for Analysis With DNA Amplification
low pH after alkaline treatment, forming large aggregates Serum, plasma, cerebrospinal fluid|| (0.5 mL) 0.3–3 μg
instead. As a result, they precipitate out of solution. The
relatively small plasmids return to their supercoiled state Dried blood (0.5- to 1-cm-diameter spot) 0.04–0.7 μg
and stay in solution. Alkaline lysis procedures were used
Saliva (1 mL) 5–15 μg
extensively for extraction of 1- to 50-kb plasmid DNA
from bacteria during the early days of recombinant DNA Buccal cells (1 mg) 1–10 μg
technology.
Bone, teeth (500 mg) 30–50 μg
Hair follicles# 0.1–0.2 μg

Preparing the Sample
Fixed tissue** (5–10 × 10-micron sections; 6–50 μ
Nucleic acid is routinely isolated from human, fungal, 10 mm2)
bacterial, and viral sources in the clinical laboratory
(Table 3.1). The initial steps in nucleic acid isolation Feces†† (animal cells, 1 mg) 2–100 pg
depend on the nature of the starting material and the test
method. Some test systems include sample-collection *Yields are based on optimal conditions. Assays will vary in yield and purity
of sample DNA.
vessels and reagents that begin the nucleic isolation in †
DNA yield will vary with WBC count.
transport to the laboratory. Details of sample collection ‡
DNA yield will depend on type and condition of tissue.
§
are important because of the diversity of sample types ||
Different bacterial types and fungi will yield more or less DNA.
DNA yield will depend on degree of cellularity.
and nucleic acid sources (viruses, bacteria, nucleated ¶
Dried blood yield from paper is less than from textiles.
cells). Use of an improper collection tube or method #
Mitochondrial DNA is attainable from hair shafts.
will compromise the test results. Test validation should **Isolation of DNA from fixed tissue is affected by the type of fixative used
and the age and the preliminary handling of the original specimen.
include the sample-collection methods. ††
Cells in fecal specimens are subjected to digestion and degradation.
80 Section II • Common Techniques in Molecular Biology
Viruses
Advanced Concepts
Viral DNA is held within free viruses or integrated into
In surveying the literature, especially early ref- the host genome along with host DNA. Some proce-
erences, the starting material determined which dures use cell-free specimens, such as plasma, for viral
DNA extraction procedure was used. Extraction detection. Others may require concentration of viroids
procedures were also modified to optimize the by centrifugation or other methods.
yield of specific products. A procedure designed
to yield plasmid DNA did not efficiently isolate Nucleated Cells in Suspension (Blood and Bone
chromosomal DNA and vice versa. Marrow Aspirates)
Nucleic acid in human blood or bone marrow comes
mostly from white blood cells (WBCs). Anticoagulants
Bacteria and Fungi added to the collected sample will prevent clotting,
which will trap the WBCs. Serum from clotted blood
Many of the early recombinant DNA experiments were is a rich source of proteins, lipids, and other molecules,
performed with gram-negative bacteria. Methods used at but not nucleic acids. Free WBCs carrying nucleic acids
that time are the basis for a variety of DNA isolation and cell-free nucleic acids are available from plasma.
systems currently used. Toxic substances used in the WBCs in blood or bone marrow specimens are purified
older methods have been replaced with safer materials. of red blood cells (RBCs) and other blood components
Cell walls are not thick and can be lysed by high pH and by either differential density-gradient centrifugation or
detergents. differential lysis.
Some bacteria and fungi have tough cell walls that For differential density-gradient centrifugation,
must be broken to allow the release of nucleic acid. whole blood or bone marrow mixed with isotonic saline
Several enzyme products that digest cell wall polymers is overlaid with Ficoll. Ficoll is a highly branched
are commercially available. Alternatively, cell walls can sucrose polymer that does not penetrate biological
be broken mechanically by grinding or by vigorously membranes. Upon centrifugation, the mononuclear
mixing with glass beads. Gentler enzymatic methods WBCs (the desired cells for isolation of nucleic acid)
are less likely to damage chromosomal DNA and thus settle into a layer in the Ficoll gradient that is below
are preferred for methods involving larger chromosomal the less dense plasma components and above the poly-
targets as opposed to plasmid DNA. Treatment with morphonuclear cells and RBCs. The layer containing
detergent (1% sodium dodecyl sulfate) and strong base the mononuclear WBCs is removed from the tube and
(0.2 M NaOH) in the presence of Tris base, ethylene- washed by rounds of resuspension and centrifugation in
diaminetetraacetic acid (EDTA), and glucose can also saline before proceeding with the nucleic acid isolation
break bacterial cell walls. procedure.
Boiling in dilute sucrose, Triton X-100 detergent, Another method used to isolate nucleated cells takes
Tris buffer, and EDTA after lysozyme treatment releases advantage of the differential osmotic fragility of RBCs
DNA that can be immediately precipitated with alcohol and WBCs. Incubation of whole blood or bone marrow
(see following discussion). DNA extracted by boiling in hypotonic buffer or water will result in the lysis of
or alkaline (NaOH) procedures is denatured (single the RBCs before the WBCs. The WBCs are pelleted
stranded) and may not be suitable for methods, such as by centrifugation, leaving the empty RBC membranes
restriction enzyme analysis, that require double-stranded (ghosts) and hemoglobin, respectively, in suspension
DNA. and solution.
Commercial reagents designed for isolation of DNA
in amplification procedures, such as polymerase chain
Plasma
reaction (PCR), are used for yeast, filamentous fungi,
and gram-positive bacteria. The advantage of these types Solid tumors and transplanted organs release cells, exo-
of extraction is their speed and simplicity. somes, and nucleic acid into the bloodstream.3,4 Shed
cells have the molecular characteristics of the shed-

ding tissue or organ, as does cell-free nucleic acid. TABLE 3.2 Tissue Fixatives Influencing
Exosomes are small vesicles (30 to 100 nm in diame- Nucleic Acid Quality7
ter), which form by invagination and budding from the
inside of cellular endosome vesicles and are secreted by Relative Average Fragment
Quality of Fixative Size
living cells. They contain proteins, lipids, and nucleic Fixative Nucleic Acid Range (kb)
acid.5 They can be collected by centrifugation. With
the high sensitivity achievable from amplification pro- 10% buffered Good 2.0–5.0
cedures, these sources of circulating nucleic acids can neutral formalin
be detected for purposes of diagnostic and prognostic Acetone Good 2.0–5.0
analyses. Such a test is called a liquid biopsy. Liquid
biopsies may preclude surgical biopsies and allow Zamboni’s Not as good 0.2–2.0
serial biopsy testing. Isolation of cell-free nucleic acid Clarke’s Not as good 0.8–1.0
requires procedures to concentrate the target nucleic
acid before isolation. These procedures include solid- Paraformaldehyde Not as good 0.2–5.0
phase collection of nucleic acid on beads or columns
Methacarn Not as good 0.7–1.5
(see following discussion). Circulating tumor cells can
be collected on special columns and filters that will Formalin–alcohol– Not as good 1.0–4.0
selectively bind to tumor cells, avoiding WBCs that may acetic acid
be present. B-5 Less desirable <0.1
Carnoy’s Less desirable 0.7–1.5

Tissue Samples
Zenker ’s Less desirable 0.7–1.5
Fresh or frozen tissue samples are dissociated before
DNA isolation procedures can be started. Grinding Bouin’s Less desirable <0.1
the frozen tissue in liquid nitrogen, homogenizing the
tissue, or simply mincing the tissue using a scalpel
disrupts the whole-tissue samples. Fixed, embedded
tissue may be deparaffinized by soaking in xylene DNA Isolation Chemistries
(a mixture of three isomers of dimethylbenzene). Less
Organic Isolation Methods
toxic xylene substitutes are also often used for this
purpose. After xylene treatment, the tissue is rehydrated After release of DNA from the cell, further purification
by soaking it in decreasing concentrations of ethanol. requires removal of contaminating proteins, lipids, car-
Alternatively, fixed tissue may be used directly without bohydrates, and cell debris. For organic isolation, this is
dewaxing. accomplished using a combination of high salt, low pH,
Depending on the fixative, DNA in preserved tissue and an organic mixture of phenol and chloroform. The
may be broken and/or cross-linked to varying extents.6 combination readily dissolves hydrophobic contami-
Comparative studies have shown that buffered formalin is nants, such as lipids and lipoproteins; collects cell debris
the least damaging among tested fixatives, with mercury- and strips away most DNA-associated proteins (Fig.
based fixatives, such as Bouin’s and B-5, being the 3.1). Isolation of small amounts of DNA from challeng-
worst for DNA recovery (see Table 3.2).7,8 DNA quality ing samples such as fungi can be facilitated by pretreat-
will also depend on how the tissue was handled prior to ment with cetyltrimethylammonium bromide, a cationic
fixation and the length of time the tissue was in fixation. detergent that efficiently separates DNA from polysac-
In general, DNA target products of 100 base pairs (bp) charide contamination. To avoid RNA contamination,
or less can consistently be obtained from fixed tissue. RNase, an enzyme that degrades RNA, can be added at
Extended digestion in proteinase K may yield longer this point. Alternatively, RNase may also be added to the
fragments. resuspended DNA at the end of the procedure.
DNA in DNA
aqueous precipitation
solution (ethanol)
Lysis Acidification Extraction
(NaOH, SDS) (acetic acid, (phenol,
salt) chloroform)
Cells in
suspension Organic
Lysed cells
Cell debris phase
DNA
FIGURE 3.1 General scheme of organic DNA isolation. After lysis of cells, cellular contents are acidified, and particulate matter
is pelleted and extracted with phenol and chloroform. This emulsion will separate into two phases. The upper aqueous phase con-
tains the DNA that can be precipitated by mixing with ethanol. Collecting the precipitated DNA by centrifugation and resuspending
it in a small volume of buffer or water (50 to 250 μL) increases the DNA concentration of the isolated DNA.
A biphasic emulsion forms when phenol and chloro-

form are added to the hydrophilic suspension of lysed ethanol cannot be distilled. The isopropanol used
cells (lysate). At the proper pH, centrifugation will settle is undenatured, or pure, because it is composed
the hydrophobic phase on the bottom, with the hydro- of 99% isopropanol and 1% water, with no other
philic phase on top. Lipids and other hydrophobic com- components.
ponents will dissolve in the lower hydrophobic phase. The choice of which alcohol to use depends on
DNA will dissolve in the upper aqueous phase. Amphi- the starting material, the size and amount of DNA
philic components, which have both hydrophobic and to be isolated, and the design of the method. Iso-
hydrophilic properties and cell debris, will collect as a propanol is less volatile than ethanol and precip-
white precipitate at the interface between the two layers. itates DNA at room temperature. Precipitation at
The DNA in the upper phase is then precipitated by room temperature reduces coprecipitation of salt.
mixing with ethanol or isopropanol and salt (ammo- Also, compared with ethanol, less isopropanol is
nium, potassium or sodium acetate, or lithium or sodium added for precipitation; therefore, isopropanol can
chloride). The ethyl or isopropyl alcohol is added to the be more practical for large-volume samples. For
upper-phase solution at 2:1 or 1:1 ratios, respectively, low concentrations of DNA, longer precipitation
and the DNA forms a solid precipitate. times at freezer temperatures may be required to
maximize the amount of DNA that is recovered.
An important consideration to precipitating the
DNA at freezer temperatures is that the increased
viscosity of the alcohol at low temperatures will
Advanced Concepts require longer centrifugation times to pellet the
DNA.
Both ethanol and isopropanol are used for molec-
ular applications. The ethanol is one of the gen-
eral-use formulas, reagent grade. Reagent-grade
alcohol (90.25% ethanol, 4.75% methanol, 5% Recovery of minimal amounts of DNA can be opti-
isopropanol) is denatured; that is, the ethanol is mized using carrier molecules. In early studies, yeast
mixed with other components because pure 100% RNA or glycogen was used to coprecipitate low con-
centrations of DNA.9 More recently, glycogen or linear
polyacrylamide have been used for this purpose. Gly-

cogen is a biological material derived from mussels Advanced Concepts
(e.g., Mytilus edulis). As a result, some preparations of
glycogen may contain DNA.10 Commercial preparations The presence of the chelating agent, EDTA, pro-
may be treated with DNase to avoid nucleic acid con- tects the DNA from damage by DNases present
tamination. Commercial glycogen may also include a in the environment of DNA preparations intended
color indicator to aid in detecting small pellets. Gener- for long-term storage. EDTA is a component of
ally, 10 to 20 micrograms of carrier is added per mil- TE buffer (10 mM Tris, 1 mM EDTA) and other
liliter of isopropanol mixture before incubation and resuspension buffers. The EDTA will also inhibit
centrifugation. enzyme activity when the DNA is used in various
The DNA precipitate is collected by centrifugation. procedures, such as restriction enzyme digestion or
Excess salt is removed by rinsing the pelleted nucleic PCR. One must be careful not to dilute the DNA
acid in 70% ethanol, centrifuging, and discarding the too far so that large volumes (e.g., more than 10%
supernatant, then dissolving the DNA pellet in rehy- of a reaction volume) of the DNA–EDTA solu-
dration buffer, usually 10 mM Tris, 1 mM EDTA (TE), tion are required for subsequent analysis methods.
or water. When DNA yield is low, as is the case with some
clinical samples, it is better to dissolve it in water.
Inorganic Isolation Methods More of this can be used in analysis procedures
without adding excess amounts of EDTA. Because
Safety concerns in the clinical laboratory make the most of or the entire sample will be used for anal-
use of caustic reagents such as phenol undesirable. ysis, protection in storage is not a concern.
Methods of DNA isolation that do not require phenol
extraction have therefore been developed and are used
in many laboratories. Initially, these methods did not
provide the efficient recovery of clean DNA achieved
with phenol extraction; however, newer methods have Advanced Concepts
proven to produce high-quality DNA preparations in
good yields. Precipitation of the DNA excludes hydrophilic
Inorganic DNA extraction is sometimes called proteins, carbohydrates, and other residual con-
“salting out” (Fig. 3.2). It makes use of low-pH and taminants still present after protein extraction. In
high-salt conditions to selectively precipitate proteins, addition, the concentration of the DNA can be
leaving the DNA in solution. The DNA can then be pre- controlled by adjusting the buffer or water volume
cipitated as described previously using isopropanol, pel- used for resuspension of the pellet.
leted and resuspended in TE buffer or water.
DNA in DNA
solution (isopropanol)
FIGURE 3.2 Inorganic DNA isola-
tion does not include the organic Lysis Protein
extraction step. Proteins are precipi- (Tris, EDTA, precipitation
tated from the cell lysate with high Cells in
SDS) (sodium
salt concentration and low pH. The suspension
acetate)
Lysed cells
supernatant containing the DNA is
then mixed with ethanol or isopropa- Protein
nol to precipitate the DNA to be col-
lected and resuspended in a smaller
volume. DNA
Solid-Phase Isolation
Advanced Concepts
More rapid and comparably effective DNA extraction
can be performed using solid matrices to bind and hold Alkaline lysis can be used to specifically select for
the DNA for washing. Silica-based products were shown plasmid DNA because chromosomal DNA will not
to effectively bind DNA in high-salt conditions.11 Many renature properly upon neutralization and form a
variations on this procedure were developed, including precipitate. The denatured chromosomal DNA and
the use of diatomaceous earth as a source of silica par- protein can be removed by centrifugation before
ticles.12 Modern systems can be purchased with solid the supernatant containing plasmid DNA is applied
matrices in the form of columns or beads. Columns come to the column.
in various sizes, depending on the amount of DNA to be
isolated. Columns used in the clinical laboratory are most
often small “spin columns” that fit inside microcentrifuge
tubes. These columns are commonly used to isolate viral Advanced Concepts
and bacterial DNA from serum, plasma, or cerebrospinal
fluid. They are also used routinely for isolation of cellular Solid matrices conjugated to specific sequences of
DNA in genetics and oncology. Preparation of samples nucleic acid can also be used to select for DNA
for isolation of DNA on solid-phase media starts with containing complementary sequences by hybrid-
cell lysis and release of nucleic acids, similar to organic ization. After removal of noncomplementary
and inorganic procedures (Fig. 3.3). Specific buffers are DNA, the bound complementary DNA can be
used to lyse bacterial, fungal, or animal cells. Buffer eluted by heating the matrix or by breaking the
systems designed for specific applications (e.g., bacterial hydrogen bonds chemically.
cell lysis or human cell lysis) are commercially available.
DNA in
aqueous
solution
DNA adsorption
Lysis Acidification (low pH)
(supplied (supplied
reagents) reagents)
Cells in
suspension
Lysed cells
Cell debris
Wash DNA Elute DNA
(supplied (low salt)
buffer)
DNA
FIGURE 3.3 Isolation of DNA on solid media. Before applying to the silicate column, particulates from the cell debris may be
removed by centrifugation. Solid-phase systems include the proprietary buffers and solutions for adhering the DNA on the column,
washing it, and eluting it in a small volume (10 to 100 μL).
For solid-phase separation, the cell lysate is applied

to a column in high-salt buffer, and the DNA in solu-
tion adsorbs to the solid matrix. Carrier RNA or DNA
is applied with the DNA to enhance recovery, prevent-
ing irreversible binding of the small amount of target
DNA. The carrier must be a nucleic acid of more than
200 nucleotides in length in order to bind to the silica
membrane. Other carriers used to enhance precipitation,
such as glycogen and linear polyacrylamide, cannot be
used in this application. After the immobilized DNA
is washed with buffer, the DNA is eluted in a spe-
cific volume of water, TE, or another low-salt buffer.
The washing solutions and the eluant can be drawn
through the column by gravity, vacuum, or centrifugal
force. DNA absorbed to magnetic beads is washed by FIGURE 3.4 Automated DNA isolation systems use car-
suspension of the beads in buffer and collection of the tridges containing lysis, adsorption, and elution agents and
beads using a magnet applied to the outside of the tube magnetic beads. Bar coding of reagents and sample tubes
while the buffer is aspirated or poured off. The DNA IQ assures accurate sample and reagent lot tracking.
system (Promega) uses a magnetic resin that holds a spe-
cific amount of DNA (100 ng). When the DNA is eluted
in 100 μL, the DNA concentration is known (1 ng/μL) either preclude or prohibit extensive DNA purification.
and ready for analysis. These include screening large numbers of samples by
Solid-phase isolation is the methodology employed simple methods (e.g., electrophoresis with or without
for most robotic DNA isolation systems that use restriction enzyme digestion and some amplification
magnetized glass beads or membranes to bind DNA procedures), isolation of DNA from limited amounts of
(Fig. 3.4). These systems are finding increased use in starting material, and isolation of DNA from challeng-
clinical laboratories for the automated isolation of DNA ing samples such as fixed, paraffin-embedded tissues.
from cultures, blood, tissue, bone marrow, plasma, and In these cases, simple lysis of cellular material in the
other body fluids. DNA isolated from specimens, such sample will yield sufficiently useful DNA for amplifica-
as urine and amniotic fluid, has been reported to work tion procedures.
better in PCR applications, possibly due to the removal Simple screening methods are mostly used for
of the inhibitory substances found in these types of research purposes in which large numbers of samples
specimens by the prefiltering step. In most systems, must be processed. In contrast, analysis of paraffin
a measured amount of sample—for example, 200 to samples is frequently performed in the clinical labo-
1,000 μL of whole blood, 100-μL cell suspensions (106 ratory. Fixed tissue is more conveniently accessed in
to 107 cells), or 10 to 50 mg of tissue, in sample tubes— the laboratory and may sometimes be the only source
is placed into the instrument along with cartridges or of patient material. Thin sections are usually used for
racks of tubes containing the reagents used for isolation. analysis, although sectioning is not necessary with
Reagents are formulated in sets depending on the type very small samples such as needle biopsies. Paraffin-
and amount of starting material. The instrument is then embedded specimens can be dewaxed with xylene or
programmed to lyse the cells and isolate and elute the other agents and then rehydrated before nucleic acid iso-
DNA automatically. lation, the fixed tissue can be used without dewaxing.
For some tests, such as somatic mutation analyses, an
adjacent stained serial section can be examined micro-
Proteolytic Lysis of Fixed Material
scopically to identify areas of the tissue comprising
Although high-quality DNA preparations are tantamount the appropriate percentage of tumor cells. The identi-
to successful procedures, there are circumstances that fiable areas of the tumor can then be isolated directly
Tissue embedded Rapid Extraction Methods

in paraffin Target cells
With the advent of PCR, new and faster methods of
DNA isolation have been developed. The minimal
sample requirements of amplification procedures allow
for the use of material previously not utilizable for anal-
Paraffin ysis. Rapid lysis methods and DNA extraction/storage
cards provide sufficiently clean DNA that can be used
for amplification.
Chelex is a cation-chelating resin that can be used
for simple extraction of DNA from minimal samples.13
A suspension of 10% Chelex resin beads is mixed with
A Microdissection the specimen, and the cells are lysed by boiling. After
Deparaffinize centrifugation of the suspension, DNA in the supernatant
(xylene, ethanol is cooled and may be further extracted with chloroform
wash)
before use in amplification procedures. This method is
most commonly used in forensic applications14,15 but
5–20 micron
may also be useful for purification of DNA from clinical
sections
samples and fixed, paraffin-embedded specimens16 and
samples spotted on storage cards.17,18
Digest
(proteinase K,
Isolation of Mitochondrial DNA
Tris buffer) There are two approaches to the isolation of mitochon-
drial DNA from eukaryotic cells. One method is to
first isolate the mitochondria by centrifugation. After
B cell preparations are homogenized by grinding on ice,
FIGURE 3.5 Crude extraction of DNA from fixed, paraf- the homogenate is centrifuged at low speed (700 to
fin-embedded tissue. Selected regions of tissue are scraped 2,600 × g) to pellet intact cells, nuclei, and cell debris.
from slides (macrodissection; A) and extracted in proteinase K The mitochondria are pelleted from the supernatant in a
(B). second high-speed centrifugation (10,000 to 16,000 × g)
and lysed with detergent. The lysate is treated with pro-
teinase to remove protein contaminants. Mitochondrial
DNA can then be precipitated with cold ethanol and
from the slide by lifting or scraping in buffer with or resuspended in water or appropriate buffers for analysis.
without the use of a microscope (microdissection or The second approach to mitochondrial DNA prepara-
macrodissection, respectively; Fig. 3.5) or laser capture tion is to isolate total DNA as described previously. The
microscopy. preparation will contain mitochondrial DNA that can be
Before lysis, cells may be washed by suspension analyzed within the total DNA background by hybrid-
and centrifugation in saline or other isotonic buffers. ization or PCR.
Reagents used for cell lysis depend on the subsequent
use of the DNA. For simple screens, cells can be lysed
in detergents, such as SDS or Triton. For use in PCR Advanced Concepts
amplification, cells may be lysed in a mixture of Tris
buffer and proteinase K. The proteinase K will digest When homogenizing cells for isolation of mito-
proteins in the sample, lysing the cells and inactivating chondria, care must be taken not to overgrind the
other enzymes. The released DNA can be used directly tissue and dissociate the mitochondrial membranes.
in the amplification reaction.
Specimen Collection
Grinding in alkaline buffers with reducing agents
such as β-mercaptoethanol will protect the mito- RNA tests, especially those that involve gene expres-
chondria during the isolation process. A high-ion- sion, such as array analysis or quantitative reverse
ic-strength buffer can also be used to selectively transcriptase PCR, are most accurate when the RNA
lyse the nuclear membranes. is stabilized from further metabolism or degradation
after collection. Specialized collection tubes have been
devised to stabilize RNA immediately upon blood draw.
In the laboratory, tissue or cell pellets can be stored and/
or transported in preservative reagents.13,19,20
ISOLATION OF RNA Advanced Concepts

Working with RNA in the laboratory requires strict pre-
Several chemical methods have been developed to
cautions to avoid sample RNA degradation. RNA is espe-
inactivate or eliminate RNases. Diethyl pyrocar-
cially labile due to the ubiquitous presence of RNases.
bonate (DEPC) can be added to water and buffers
These enzymes are small proteins that can renature,
(except for Tris buffer) to inactivate RNases per-
even after autoclaving, and regain activity. They remain
manently. DEPC converts primary and second-
active at a wide range of temperatures (down to –20°C
ary amines to carbamic acid esters. It cross-links
and below). Unlike DNases, RNases must be eliminated
RNase proteins through intermolecular covalent
or inactivated before isolation of RNA.
bonds, rendering them insoluble. Because of its
It is necessary to allocate a separate RNase-free
effect on amine groups, DEPC will adversely
(RNF) area of the laboratory for storage of materials and
affect Tris buffers. DEPC will also interact with
specimen handling intended for RNA analysis. Gloves
polystyrene and polycarbonate and is not recom-
must always be worn in the RNF area. Disposables
mended for use on these types of materials.
(tubes, tips, etc.) that come in contact with the RNA
Other RNase inhibitors include vanadyl-
should be kept at this location and never be touched by
ribonucleoside complexes, which bind the active
ungloved hands. Articles designated DNAse-free/RNF
sites of the RNase enzymes, and macaloid clays,
by suppliers may be used directly from the package.
which absorb the RNase proteins. Ribonuclease
Although reusable glassware is seldom used for RNA
inhibitor proteins can be added directly to RNA
work, it can be rendered RNF. After cleaning, glassware
preparations. These proteins form a stable nonco-
must be baked for four to six hours at 400°C to inactivate
valent complex with the RNases in solution. Some
the RNases.
of these interactions require reducing conditions;
therefore, dithiothreitol might be added in addition
Total RNA to the inhibitor.
H2 O O O CH2
There are several types of RNA in prokaryotes and
C C C C
eukaryotes. The most abundant (80% to 90%) RNA in
all cells is ribosomal RNA (rRNA). This RNA consists O O
of two components, large and small, which are visu- Diethyl pyrocarbonate.
alized by agarose gel electrophoresis (see Fig. 3.10).
Depending on the cell type and conditions, the next most
abundant RNA fraction (2.5% to 5%) is messenger RNA
RNA Isolation Chemistries
(mRNA). This mRNA may be detected as a faint back-
ground underlying the rRNA detected by agarose gel Preparation of specimen material for RNA extraction
electrophoresis. Transfer RNA and small nuclear RNAs differs in some respects from DNA extraction. Retic-
are also present in the total RNA sample. ulocytes in blood and bone marrow samples are lysed
by osmosis or separated from WBCs by centrifugation. is added at the lysis step to eliminate contamination of
When dissociating tissue, the sample should be kept DNA. Alternatively, RNF DNase also may be added
frozen in liquid nitrogen or immersed in buffer that will directly to the isolated RNA at the end of the procedure.
inactivate intracellular RNases. This is especially true After phase separation, the upper aqueous phase contain-
for tissues such as pancreas that contain large amounts ing the RNA is removed to a clean tube, and the RNA is
of innate RNases. Bacterial and fungal RNA are also iso- precipitated by addition of two volumes of ethanol or one
lated by chemical lysis or by grinding in liquid nitrogen. volume of isopropanol. Glycogen or yeast-transfer RNA
Viral RNA can be isolated directly from serum, plasma, may be added at this step as a carrier to aid RNA pellet
culture medium, or other cell-free fluids by means of formation. The RNA precipitate is then washed in 70%
specially formulated spin columns or beads. Cell-free ethanol and resuspended in RNF buffer or water.
material is used for these isolations because most total
RNA isolation methods cannot distinguish between Solid-Phase Isolation
RNA from microorganisms and those from host cells.
Solid-phase separation of RNA begins with similar
steps as described previously for organic extraction. The
Organic Isolation
strong denaturing buffer conditions must be adjusted
The cell lysis step for RNA isolation is performed in deter- before application of the lysate to the column (Fig. 3.7).
gent or phenol in the presence of high salt (0.2 to 0.5 M In some procedures, ethanol is added at this point. Some
NaCl) or RNase inhibitors. Guanidine isothiocyanate systems provide a filter column to remove particulate
(GITC) is a strong denaturant of RNases and can be used material before application to the adsorption column.
instead of high-salt buffers. Strong reducing agents such As with DNA columns, commercial reagents are sup-
as 2-mercaptoethanol may also be added during this step. plied with the columns to optimize RNA adsorption and
Once the cells are lysed, proteins can be extracted by washing on the silica-based matrix.
organic means (Fig. 3.6). Acid phenol:chloroform:iso- After lysate is applied to the column in the high-salt
amyl alcohol (25:24:1) solution efficiently extracts RNA. chaotropic buffer, the adsorbed RNA is washed with the
Chloroform enhances the extraction of the nucleic acid supplied buffers. DNase may be added directly to the
by denaturing proteins and promoting phase separation. adsorbed RNA on the column to remove contaminat-
Isoamyl alcohol prevents foaming. For RNA, the organic ing DNA. Washing solutions and the eluant are drawn
phase must be acidic (pH 4 to 6). The acidity of the through the column by gravity, vacuum, or centrifu-
organic phase is adjusted by overlaying it with a buffer of gal force. Small columns of silica-based material that
the appropriate pH. In some isolation procedures, DNase fit inside microfuge tubes (spin columns) are widely
RNA in RNA
solution) (ethanol)
Lysis Extraction
(guanidinium (phenol,
isothiocyanate) chloroform)
Cells in
suspension
Lysed cells Organic
phase
RNA
FIGURE 3.6 Organic extraction of total RNA is similar to that of DNA; however, the RNA must be protected from intracellular
and extracellular RNases. The RNA in solution is precipitated and resuspended, similar to DNA. Some procedures include treat-
ment of the RNA solution with DNase to remove any contaminating DNA.
RNA adsorption
Lysis (low pH)
(supplied
reagents)
Cells in
suspension
Lysed cells
Wash RNA Elute RNA

(supplied
buffer)
RNA
FIGURE 3.7 Isolation of RNA on a solid matrix is also similar to DNA isolation. Buffers and wash solutions are designed for the
RNA target.
used for routine nucleic acid isolation from all types spin columns for removal of genomic DNA contamina-
of specimens. The eluted RNA is usually of sufficient tion are included in some systems. Automated isolation
concentration and purity for direct use in most appli- systems also have reagent kits and cartridges designed to
cations. Automated systems like those shown in Figure isolate RNA from fixed tissue.
3.4 use magnetic beads or particles for RNA isolation as
well. Special reagent sets are available for RNA or total Isolation of polyA (Messenger) RNA
nucleic acid (RNA and DNA) on these instruments.
As previously stated, approximately 80% to 90% of total
Generally, 1 million eukaryotic cells or 10 to 50 mg
RNA is rRNA. Often the RNA required for analysis is
of tissue will yield about 10 μg of RNA. The yield of
mRNA, accounting for only about 2.5% to 5% of the
RNA from biological fluids will depend on the con-
total RNA yield. The majority of mRNA consists of
centration of microorganisms or other target molecules
mRNA from highly expressed genes. Rare or single-copy
present in the specimen (Table 3.3).
mRNA is, therefore, a very minor part of the total RNA
isolation. To enrich the yield of mRNA, especially rare
Proteolytic Lysis of Fixed Material
transcripts, protocols employing single-stranded oligo-
The quality of RNA from fixed, paraffin-embedded mers of thymine or uracil immobilized on a matrix resin
tissue will depend on the fixation process and han- column or beads are used (Fig. 3.8). The polyT or polyU
dling of the specimen before fixation (Table 3.2).21,22 oligomers will bind the polyA tail found exclusively on
Commercial reagent sets are available for extraction of mRNA. After washing away residual RNA, polyA RNA
RNA from fixed-tissue specimens. These reagents are is eluted by washing the column with warmed, low-salt
designed to provide lysis and incubation conditions that buffer containing detergent to break the hydrogen bonds
reverse formaldehyde modification of RNA and effi- between the mRNA and the column. With this approach,
ciently release RNA from tissue sections while avoid- approximately 30 to 40 ng of mRNA can be obtained
ing further RNA degradation. Specialized reagents or from 1 μg of total RNA.
oligomer. Also, mRNAs with short polyA tails may

TABLE 3.3 Yield of RNA From Various not bind efficiently or at all. AT-rich DNA fragments
Specimen Sources36-38 might bind to the column and not only compete with
the desired mRNA target but also contaminate the final
Specimen Expected Yield* eluant. Potential digestion of the oligo-conjugated matri-
Blood† (1 mL, 3.5–10 × 106 WBCs/mL) 1–10 μg ces precludes the use of DNase on the RNA before it is
bound to the column. Treatment of the eluant with RNF
†
Buffy coat (1 mL whole blood) 5–10 μg DNase is possible, but the DNase should be inactivated
Bone marrow† (1 mL) 50–200 μg
if the mRNA is to be used in procedures involving DNA
components. Furthermore, rRNA may co-purify with
Cultured cells‡ (107 cells) 50–150 μg the polyA RNA. The final product, then, is enriched in
polyA RNA but is not a pure polyA preparation.
Buccal cells (1 mg) 1–10 μg
Solid tissue§ (1 mg) 0.5–4 μg

MEASUREMENT OF NUCLEIC ACID QUALITY
||
Fixed tissue (1 mm ) 3
0.2–3 μg
AND QUANTITY
Bacterial culture¶ (0.5 mL, 0.7 10–100 μg
absorbance units) Laboratory analysis of nucleic acids produces vari-
able results, depending on the quality and quantity of
*Specimen handling especially affects RNA yield. Isolation of polyA RNA will
result in much lower yields. See text.
input material. This is an important consideration in the
†
RNA yield will depend on WBC count. medical laboratory because test results must be accu-
‡
§
RNA yield will depend on type of cells and the conditions of cell culture. rately interpreted with respect to disease pathology.
Liver, spleen, and heart tissues yield more RNA than brain, lung, ovary,
kidney, or thymus tissues.
Consistent results require that run-to-run variation be
||
Isolation of RNA from fixed tissue is especially affected by the type of fixative minimized. Fortunately, measurement of the quality and
used and the age and the preliminary handling of the original specimen.
¶
quantity of DNA and RNA is straightforward.
Different bacterial types and fungi will yield more or less RNA.
Electrophoresis
DNA and RNA can be analyzed for quality by resolv-
ing an aliquot of the isolated sample on an agarose gel
mRNA (Fig. 3.9). Fluorescent dyes such as ethidium bromide or
5′ A A A A A A A A A 3′ SybrGreen I bind specifically to DNA and are used to
3′ T T T T T T T T T 5′
visualize the sample preparation. Ethidium bromide or
SybrGreen II are used to detect RNA. Less frequently,
silver stain has been used to detect small amounts of
Bead or column DNA by visual inspection.
FIGURE 3.8 Oligo polythymine (or polyuracil) columns or The appearance of DNA on agarose gels depends
beads bind the polyA tail of mRNA. Peptide nucleic acid dT or on the type of DNA isolated. A good preparation of
dU can also be used. These polymers are less subject to degra- plasmid DNA will yield a bright signal from supercoiled
dation by contaminating nucleases. plasmid DNA with minor or no other bands that rep-
resent nicked or broken plasmid (see Fig. 3.10). High-
molecular-weight chromosomal DNA should collect as a
There are limitations to the isolation of polyA RNA bright band near the top of the gel. A high-quality prepa-
using oligo dT or dU. The efficiency of polyA and ration of RNA will yield two distinct bands of ribosomal
polyU binding is variable. Secondary structure (intra- RNA. The integrity of these bands is an indication of the
strand or interstrand hydrogen bonds) in the target integrity of the other RNA species present in the same
sample may compete with binding to the capture sample. If these bands are degraded (smeared) or absent,
L N, SC
M SC N L Nicked/relaxed
23 kb
Linear
0.6 kb Supercoiled
FIGURE 3.9 After agarose gel electrophoresis, compact supercoiled plasmid DNA (SC) will travel farther through the gel than
nicked plasmid (N), which has single-strand breaks. Relaxed plasmid DNA (R) has double-strand breaks and will migrate accord-
ing to its size, 23 kb in the drawing on the left. Linear (L) plasmids migrate according to the size of the plasmid. A gel photo shows
a plasmid preparation. N, nicked; SC, supercoiled; L, linear; R, relaxed; M, molecular-weight markers.
Wells
Genomic
DNA
28S rRNA
18S rRNA
FIGURE 3.10 Intact ethidium bromide–stained human chromosomal DNA (left) and total RNA (right) after agarose gel electro-
phoresis. High-quality genomic DNA runs as a tight smear close to the loading wells. High-quality total RNA appears as two rRNA
bands representing large and small ribosomal RNA species (shown with molecular weight markers, M).
the quality of the RNA in the sample is deemed unac- with that of a known amount of control DNA or RNA
ceptable for use in molecular assays. loaded on the same gel. Densitometry of the band inten-
When fluorescent dyes are used, DNA and, less accu- sities gives the most accurate measurement of quantity.
rately, RNA can be quantified by comparison of the flu- For some procedures, estimation of DNA or RNA quan-
orescence intensity of the sample aliquot run on the gel tity can be made by visual inspection.
Spectrophotometry
EXAMPLE 2: An RNA preparation diluted 1/10
Nucleic acids absorb light at 260 nm through the adenine
yields an absorbance reading of 0.500 at 260 nm.
residues. Using the Beer–Lambert law, concentration
The concentration is
can be determined from the absorptivity constants
(50 for double-stranded DNA, 40 for RNA). The rela- 0.500 absorbance units × 40 μg mL
tionship of concentration to absorbance is expressed as per absorbance unit × 10 = 200 μg mL
A = ∈bc The yield of the sample is calculated using the
where A = absorbance, ∈ = molar absorptivity (L/mol- volume of the preparation. If the DNA was eluted
cm), b = path length (cm), and c = concentration (mg/L). or resuspended in 0.2 mL in the case illustrated
The absorbance at this wavelength is thus directly previously, the yield would be
proportional to the concentration of the nucleic acid 200 μg mL × 0.2 mL = 40 μg
in the sample. Using the absorptivity as a conversion
factor from optical density to concentration, one optical
density unit (or absorbance unit) at 260 nm is equivalent
to 50 mg/L (or 50 μg/mL) of double-stranded DNA and
40 μg/mL of RNA. To determine concentration, multiply Histooricaal Higghlligghtts
the spectrophotometer reading in absorbance units by
the appropriate conversion factor. Phenol absorbs ultra- The maximum absorption of light at 260 nm was
violet light at 270 to 275 nm, close to the wavelength of one of the clues suggesting that DNA was the
maximum absorption by nucleic acids. This means that molecular matter of genetic material. In 1942,
residual phenol from organic isolation procedures can Lewis Stadler reported results on studies of the
increase 260 readings, so phenol contamination must be effect of wavelength of ultraviolet (UV) light on
avoided when measuring concentration at 260 nm. mutagenesis in corn plants.23 He observed that
If the DNA or RNA preparation is of sufficient con- the most mutagenic monochromatic light had a
centration to require dilution before spectrophotometry wavelength of 260 nm. Together with the data of
in order for the reading to fall within the linear reading Avery24 and observations of Hershey and Chase,25
range, the dilution factor must be included in the calcu- Stadler ’s observations further supported the idea
lation of quantity. Multiply the absorbance reading by that DNA is the carrier of genetic information.
the conversion factor and the dilution factor to find the
concentration of nucleic acid.
Spectrophotometric measurements may also be used to

EXAMPLE 1: A DNA preparation diluted /100 1 estimate the purity of nucleic acid. Ideally, contamina-
yields an absorbance reading of 0.200 at 260 nm. tion can be detected by reading the concentration over a
To obtain the concentration in μg/mL, multiply: range of wavelengths. Graphing the absorbance reading
as a function of wavelength produces a curve or spec-
0.200 absorbance units × 50 μg mL trum that should peak at A260 nm for nucleic acid. Some
per absorbance unit × 100 = 1, 000 μgg mL spectrophotometers produce this graph automatically.
The yield of the sample is calculated using the Even with such automation, however, spectral analy-
volume of the preparation. If the DNA was eluted sis for each specimen sample is not practical for most
or resuspended in a volume of 0.5 mL in the case routine medical laboratory work. In practice, nucleic
illustrated previously, the yield would be acid solutions are read at two or three distinct wave-
lengths (Table 3.4). Absorbance over background at any
1, 000 μg mL × 0.5 mL = 500 μg wavelength other than the A260 maxima of the nucleic
acid indicates contamination.
fragmented or degraded DNA will produce an A260

TABLE 3.4 Common Contaminants and Their reading comparable to intact DNA. In this regard, fluo-
Wavelengths of Peak Absorbance rometry is considered by some to be a better method of
quality assessment.
Wavelength (nm) Contaminant
230 Organic compounds Histooricaal Higghlligghtts

270 Phenol
Estimation of protein contamination in nucleic
280 Protein acid is based on a method by Warburg and Chris-
tian designed to measure nucleic acid contamina-
>330 Particulate matter tion in protein.26 This method utilizes the natural
light absorption at 280 nm of the aromatic amino
acid side chains, tryptophan, and tyrosine. Nucleic
Routine nucleic acid isolation procedures produce acids are common contaminants of protein prepa-
reasonably pure DNA solutions free from particles, rations and naturally absorb light at 260 nm,
organic compounds, and phenol. Due to its abundance where there is no interference by protein. In the
and close association with nucleic acid in the cell, the Warburg–Christian method, the test material was
most likely contaminant in a nucleic acid preparation read at 260 and 280 nm, and then a nomograph
will be protein. Protein absorbs light at 280 nm through was used to determine the relative amounts of
the aromatic tryptophan and tyrosine residues. Although protein and nucleic acid.
this measurement is not highly accurate, general ranges
indicate at least the presence of protein contaminants.
The absorbance of the nucleic acid at 260 nm should be
1.6 to 2.00 times more than the absorbance at 280 nm. Advanced Concepts
If the 260-nm/280-nm ratio is less than 1.6, the nucleic
acid preparation may be contaminated with unaccept- Ultraviolet spectrophotometers dedicated to
able amounts of protein and not of sufficient purity for nucleic acid analysis can be programmed to cal-
use. Such a sample can be improved by column purifi- culate absorbance ratios and concentrations. The
cation, reprecipitating the nucleic acid, or repeating the operator must enter the type of nucleic acid, the
protein-removal step of the isolation procedure. It should dilution factor, and the desired conversion factor.
be noted that low pH affects the 260-nm/280-nm ratio. The instrument will automatically read the sample
Somewhat alkaline buffers (pH 7.5) are recommended at the appropriate wavelengths and do the required
for accurate determination of purity. RNA affords a calculations, giving a reading of concentration in
somewhat higher 260-nm/280-nm ratio, 2.0 to 2.3. A μg/mL and a 260-nm/280-nm ratio.
DNA preparation with a ratio higher than 2.0 may be
contaminated with RNA. Some procedures for DNA
analysis are not affected by contaminating RNA, in
Fluorometry
which case the DNA is still suitable for use. If, however,
RNA may interfere or react with DNA-detection compo- Fluorometry, or fluorescent spectroscopy, measures flu-
nents, RNase should be used to remove the contaminat- orescence related to DNA concentration in association
ing RNA. Because it is difficult to detect contaminating with DNA-specific fluorescent dyes. Early methods
DNA in RNA preparations, RNA should be treated with used 3,5-diaminobenzoic acid 2HCl (DABA).27 This
RNF DNase where DNA contamination may interfere dye combines with alpha-methylene aldehydes (deoxy-
with subsequent applications. ribose) to yield a fluorescent product. It is still used for
Absorbance ratios do not necessarily predict success- the detection of nuclei and as a control for hybridiza-
ful amplification or hybridization reactions. Because tion and spot integrity in microarray analyses. Although
the light is absorbed by single-nitrogen-base moieties, less convenient to use, fluorometry is recommended for
procedures where accurate measurement of intact DNA Absorption and fluorometry readings may not always
is critical, such as next-generation sequencing. agree. First, the two detection methods recognize differ-
More modern procedures use the DNA-specific dye ent targets. Single nucleotides do not bind to fluorescent
Hoechst 33258. This dye combines with adenine-thymine dyes, but they can absorb ultraviolet light and affect
base pairs in the minor groove of the DNA double helix spectrometric readings. Furthermore, absorption mea-
and is thus specific for intact double-stranded DNA. The surements do not distinguish between DNA and RNA,
binding specificity for A-T residues, however, complicates so contaminating RNA may be factored into the DNA
measurements of DNA that have unusually high or low GC measurement. RNA does not enhance the fluorescence
content. A standard measurement is required to determine of the fluorescent dyes and is thus invisible to fluoro-
concentration by fluorometry, and this standard must have metric detection. In fact, specific detection of RNA in
GC content similar to that of the samples being measured. the presence of DNA in solution is not yet possible.
Calf thymus DNA (GC content = 50%) is often used as a Deciding which instrument to use is at the discretion
standard for specimens with unknown DNA GC content. of the laboratory. Most laboratories use spectrophotom-
Fluorometric determination of DNA concentration using etry because the samples can be read directly without
Hoechst dye is very sensitive (down to 200 ng DNA/mL). staining or mixing with dye. For methods that require
PicoGreen and OliGreen are other DNA-specific dyes accurate measurements of low amounts of DNA or RNA
that can be used for fluorometric quantification. Due to (in the range of 10 to 100 ng/mL), fluorometry may
brighter fluorescence upon binding to double-stranded be preferred.
DNA, PicoGreen is more sensitive than Hoechst dye
(detection down to 25 pg/mL concentrations). Like
Microfluidics
Hoechst dye, single-stranded DNA and RNA do not
bind to PicoGreen. OliGreen is designed to bind to short Lab-on-a-chip technology has been applied to nucleic
pieces of single-stranded DNA (oligonucleotides). This acid quantification and analysis using microfluidics-
dye will detect down to 100 pg/mL of single-stranded based instruments. To make a measurement, the sample
DNA. OliGreen will not fluoresce when bound to is applied to a multiwell chip. The sample then moves
double-stranded DNA or RNA. through microchannels across a detector. The instru-
RNA may be measured in solution using SybrGreen ment software generates images in electropherogram
II RNA gel stain.28 The intensity of SyBrGreen II flu- (peak) or gel (band) configurations. For RNA, a quan-
orescence is 20% to 26% lower with polyadenylated tification estimate called the RNA integrity number is
RNA than with total RNA. The sensitivity of this dye determined as a standard measure of RNA integrity. The
is 2 ng/mL. SybrGreen II, however, is not specific to presence of ribosomal impurities in mRNA preparations
RNA and will bind and fluoresce with double-stranded may also be calculated. This system is more automated
DNA as well. Contaminating DNA must therefore be than standard spectrophotometry or fluorometry, uses a
removed in order to get an accurate determination of minimal volume of sample (as low as 1 μL), and can
RNA concentration. test multiple samples simultaneously. Microfluidics
Fluorometry measurements require calibration of the is useful for the analysis of studies on small RNAs,
instrument with a known amount of standard before mea- such as microRNAs in eukaryotes and gene expression
surement of the sample. For methods using Hoechst dye, in bacteria.
the dye, diluted to a working concentration of 1 μg/mL
in water, is mixed with the sample (usually a dilution
of the sample). Once the dye and sample solution are
mixed, fluorescence must be read within two hours STUDY QUESTIONS
because the dye/sample complex is stable only for
approximately this amount of time. The fluorescence is
DNA Quantity/Quality
read in a quartz cuvette. A programmed fluorometer will
read out a concentration based on the known standard 1. Calculate the DNA concentration in μg/mL from the
calibration. following information:
a. Absorbance reading at 260 nm from a 2. If the volume of the RNA solutions in question 1
1:100 dilution = 0.307 was 0.5 mL, calculate the yield for (a)–(d).
b. Absorbance reading at 260 nm from a
1:50 dilution = 0.307 3. An RNA preparation has the following absorbance
c. Absorbance reading at 260 nm from a readings:
1:100 dilution = 0.172
A260 = 0.208
d. Absorbance reading at 260 nm from a
A280 = 0.096
1:100 dilution = 0.088
Is this RNA preparation satisfactory for use?
2. If the volume of the DNA solutions in question 1
was 0.5 mL, calculate the yield for (a)–(d).
4. A blood sample was held at room temperature for
5 days before being processed for RNA isolation.
3. Three DNA preparations have the following A260 and
Will this sample likely yield optimal RNA?
A280 readings:
5. Name three factors that will affect the yield of RNA
Sample OD260 OD280 from a paraffin-embedded tissue sample.
(i) 1 0.419 0.230
(ii) 2 0.258 0.225
References
(iii) 3 0.398 0.174
1. Mirsky A. The discovery of DNA. Scientific American 1968;218:
For each sample, based on the A260/A280 ratio, is each 78–88.
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contaminating the DNA? 671–682.
3. Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating liquid
4. After agarose gel electrophoresis, a 0.5-microgram biopsies into the management of cancer. Nature Reviews: Clinical
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Collins JP, Thompson K, Song M, Wang YS, Ross D, Nelles MJ,
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Validation of a clinical-grade assay to measure donor-derived cell-
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concentration by spectrophotometry with analysis by lar Diagnostics 2016;18:890–902.
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B, Zheng Y, Hoshino A, Brazier H, Xiang J, Williams C, Rodri-
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nejad N, Manova-Todorova K, Welte K, Bromberg J, Peinado H,
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following information: Carrillo-Ávila JA, López-Guerrero JA, Aguilar-Quesada R. DNA
source selection for downstream applications based on DNA quality
a. Absorbance reading at 260 nm from a indicators analysis. Biopreservation and Biobanking 2016;14:
1:100 dilution = 0.307 264–270.
b. Absorbance reading at 260 nm from a 7. Greer C, Peterson SL, Kivat NB, Manos MM. PCR amplification
1:50 dilution = 0.307 from paraffin-embedded tissues. American Journal of Clinical
c. Absorbance reading at 260 nm from a Pathology 1991;95:117–124.
8. Perry C, Chung JY, Ylaya K, Choi CH, Simpson A, Matsumoto
1:100 dilution = 0.172 KT, Smith WA, Hewitt SM. A buffered alcohol-based fixative for
d. Absorbance reading at 260 nm from a histomorphologic and molecular applications. Journal of Histo-
1:100 dilution = 0.088 chemistry and Cytochemistry 2016;64:425–440.
9. Gallagher M, Burke WF Jr, Orzech K. Carrier RNA enhancement 25. Hershey AD, Chase M. Independent function of viral protein and
of recovery of DNA from dilute solutions. Biochemical Biophysi- nucleic acid in growth of bacteriophage. Journal of General Phys-
cal Research Communications 1987;144:271–276. iology 1952;26:36–56.
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KK, Magnus P, Stoltenberg C, Susser E, Lipkin WI. Human blood 35. McOrist A, Jackson M, Bird AR. A comparison of five methods of
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Chapter 4
Resolution and Detection
of Nucleic Acids
Outline Objectives
ELECTROPHORESIS OF NUCLEIC ACIDS 4.1 Explain the principle and performance of
GEL SYSTEMS electrophoresis as it applies to nucleic acids.
Agarose Gels 4.2 Compare and contrast the agarose and
Pulsed-Field Gel Electrophoresis polyacrylamide gel polymers commonly used to
Polyacrylamide Gels resolve nucleic acids, and state the utility of each
CAPILLARY ELECTROPHORESIS polymer.
BUFFER SYSTEMS 4.3 Explain the principle and performance of capillary
Buffer Additives electrophoresis as applied to nucleic acid
ELECTROPHORESIS EQUIPMENT separation.
Gel Loading 4.4 Give an overview of buffers and buffer additives
DETECTION SYSTEMS used in electrophoretic separation, including the
Fluorescent Dyes constituents, purpose, and importance.
Intercalating Agents 4.5 Describe the general types of equipment used for
Minor Groove–Binding Dyes electrophoresis and how samples are introduced for
Silver Stain electrophoretic separation.
4.6 Compare and contrast pulsed-field gel
electrophoresis and regular electrophoresis
techniques with regard to method and applications.
4.7 Describe detection systems used in nucleic acid
applications.
97
Resolution and detection of nucleic acids are done in

several ways. Gel and capillary electrophoresis are Advanced Concepts
the most practical and frequently used methods. DNA
can also be spotted and detected using specific hybrid- Double-stranded DNA and RNA are analyzed
ization probes, as is described in Chapter 9, “DNA by native gel electrophoresis. The relationship
Sequencing.” between size and speed of migration can be
improved by separating single-stranded nucleic
acids; however, both DNA and RNA favor the
ELECTROPHORESIS OF NUCLEIC ACIDS double-stranded state. When complementary
strands are not available, single strands will fold
Electrophoresis is the movement of molecules by an and form hydrogen bonds, forming hairpin-like
electric current. This can occur in air or solution, or in a structures, and imperfectly complementary strands
matrix to limit migration and contain the migrating mate- will form heteroduplexes. This intrastrand or
rial. Electrophoresis is routinely applied to the analysis imperfect interstrand hydrogen bonding will
of proteins and nucleic acids. Each phosphate group on impair migration. Unpaired, or denatured, DNA
a nucleic acid polymer is ionized, making the molecule and RNA must therefore be analyzed in condi-
negatively charged. Under an electric current, DNA and tions that prevent the hydrogen bonding between
RNA will migrate toward the positive pole (anode). In a complementary sequences. These conditions are
matrix of agarose or polyacrylamide, migration under maintained through a combination of formamide
the pull of the current is impeded, depending on the mixed with the sample, urea mixed with the gel,
size of the molecules and the spaces in the gel matrix. and/or heat.
Because each nucleotide has one negative charge, the
charge-to-mass ratio of molecules of different sizes will
remain constant. DNA and RNA will therefore migrate
at speeds inversely related to the size or length of the
polymer. Electrophoresis is performed in tubes, slab
gels, or capillaries. Slab gel electrophoresis has either a Histooricaal Higghlligghtts
horizontal or vertical format (Fig. 4.1).
Arne Tiselius developed an electrophoresis appa-
ratus while studying resolution diffusion and
adsorption of proteins in naturally occurring
porous silicate particles. This “moving-boundary”
– method required equipment over 5 m long. By the
early 1930s, his method was further refined.1 In
the early 1950s, granular starch was introduced
as a matrix.2 Oliver Smithies later found that if he
heated about 15 grams of starch in 100 mL of his
electrophoretic medium and allowed it to cool, he
could make a gel matrix with resolving properties
– +
similar to a paper electrophoresis method he had
previously devised. He first used this gel to sepa-
rate serum proteins.3 In 1956, Smithies and Poulik
+ described the resolution of 20 serum protein com-
FIGURE 4.1 Horizontal (left) and vertical (right) gel electro- ponents using a two-dimensional electrophoresis
phoresis. In both formats, the sample is introduced into the gel system with paper in one dimension and starch
at the cathode end (small arrows) and migrates with the current in the other.4
toward the anode.
Chapter 4 • Resolution and Detection of Nucleic Acids 99
GEL SYSTEMS TABLE 4.1 Choice of Agarose Concentration

for DNA Gels*14
Gel matrices provide resistance to the movement of
molecules under the force of an electric current. They
Separation Range
prevent diffusion and reduce convection currents so Agarose Concentration (%) (size in bp)
that the separated molecules form a defined group, or
“band.” The gel can then serve as a support medium 0.3 5,000–60,000
for analysis of the separated components. The best
0.6 1,000–20,000
matrix would be unaffected by electrophoresis, simple
to prepare, and amenable to modification. Agarose and 0.8 800–10,000
polyacrylamide are polymers that meet these criteria.
1.0 400–8,000
1.2 300–7,000
Agarose Gels
1.5 200–4,000
Agarose is a polysaccharide polymer extracted from
seaweed. It is a component of agar used in bacterial 2.0 100–3,000
culture dishes, which is comprised of agaropectin and
agarose. Agarose is a linear polymer of agarobiose, *The table shows the range of separation for linear double-stranded DNA
which consists of 1,3-linked-β-d-galactopyranose and molecules in TAE agarose gels with regular power sources. Note that these
values may be affected if another running buffer is used and if the voltage is
1,4-linked 3, 6-anhydro-α-l-galactopyranose (Fig. 4.2). over 5 V/cm.
Hydrated agarose gels in various concentrations,
buffers, and sizes can be purchased ready for use. Alter-
natively, agarose is purchased and stored in the labora- very low concentrations produce a weak gel that is
tory in powdered form. For use, powdered agarose is easily broken. The gel strength of any concentration of
suspended in buffer, heated, and poured into a mold. agarose will also decrease over time and with exposure
The concentration of the agarose dictates the size of to chaotropic agents such as urea.
the spaces in the gel (100 to 300 nm). The size of DNA
to be resolved is considered in determining the appro-
priate agarose concentration to use (Table 4.1). Small Advanced Concepts
pieces of DNA (50 to 500 base pairs [bp]) are resolved
on more concentrated agarose gels, for example, 2% The physical characteristics of the agarose gel
to 3% (Fig. 4.3). Larger fragments of DNA (2,000 to can be modified by altering its polymer length
50,000) are best resolved in lower agarose concentra- and helical parameters. Several types of agarose
tions, for example, 0.5% to 1%. Agarose concentrations are thus available for specific applications. The
above 5% and below 0.5% are not practical. High- resolving properties differ in these preparations as
concentration agarose will impede migration, whereas well as the gelling properties. Low-melting-point
agarose is often used for re-isolating resolved
fragments from the gel. Other agarose types give
O better resolution of larger or smaller fragments.
6 CH2OH
5 O 3 2 Agarose preparations are sufficiently pure to avoid
OH O OH problems such as electroendosmosis, a solvent
OH 1␣
4
1␤ 4
6 CH2 flow toward one of the electrodes, usually the
O cathode (negative), in opposition to the DNA
O 2 5 O
3
or RNA migration, which slows and distorts the
OH migration of the samples, reducing resolution and
FIGURE 4.2 Agarobiose is the repeating unit of the agarose smearing the bands.
polymer.
2% 4% 5% FIGE TAFE
–
–
+
Above
gel
500 bp Below
gel
+
500 bp +
RGE CHEF
200 bp
– –
200 bp
– +
800 bp
+
+
FIGURE 4.4 Field-inversion gel electrophoresis (FIGE),

transverse alternating-field electrophoresis (TAFE), rotating
500 bp
gel electrophoresis (RGE), and contour-clamped homogeneous
electric field (CHEF) are all examples of pulsed-field gel con-
50 bp figurations. Arrows indicate the migration paths of the DNA.
For these very large DNA molecules, pulses of current

200 bp applied to the gel in alternating dimensions enhance
migration. This process is called pulsed-field gel elec-
50 bp trophoresis (PFGE) (Fig. 4.4). The simplest approach
to this method is field-inversion gel electrophoresis
(FIGE).5 FIGE works by alternating the positive and
50 bp negative electrodes during electrophoresis. In this type
of separation, the DNA goes periodically forward and
backward. FIGE requires temperature control and a
FIGURE 4.3 Resolution of double-stranded DNA fragments switching mechanism. Field inversion or reorientation
on 2%, 4%, and 5% agarose. As the gel concentration increases, to move very large molecules is performed in other
the movement of particles (DNA fragments) slows. configurations. Contour-clamped homogeneous electric
field,6 transverse alternating-field electrophoresis,7 and
rotating gel electrophoresis8,9 are examples of transverse
Pulsed-Field Gel Electrophoresis
angle-reorientation electrophoresis methods.
Very large pieces (50,000 to 250,000+ bp) of DNA Field-inversion/reorientation systems require gel box,
cannot be resolved efficiently by simple agarose electro- electrode, and gel configurations as well as appropriate
phoresis. Even in the lowest concentrations of agarose, electronic control to accommodate switching the elec-
megabase fragments are too severely impeded for correct tric fields during electrophoresis. Using various forms
resolution (referred to as limiting mobility). Limiting of PFGE, the large fragments are resolved, not only
mobility is reached when a DNA molecule is of such a by sifting through the spaces in the polymer but also
size that it can move only lengthwise through successive by their reorientation. PFGE methods require long sep-
pores of the gel. aration times because the electrophoresis requires low
voltage and the time necessary for the DNA molecules the plug is inserted directly into a space or well in the
to realign themselves to move in a second dimension, agarose gel for electrophoresis. PFGE instruments will
usually an angle of 120° (180° for FIGE) from the orig- then apply current in alternating directions at specific
inal direction of migration. times (called the switch interval) that are set by the
operator. These parameters are based on the general size
of the fragments to be analyzed; that is, a larger frag-
Advanced Concepts ment will require a longer switch interval. PFGE is a
slow migration method. Sample runs will take 24 hours
FIGE is a special modification of PFGE in which or more.
the alternating currents are aligned 180° with Alternating-field electrophoresis is used for applica-
respect to each other. The current pulses must tions that require the resolution of chromosome-sized
be applied at different strengths and/or durations fragments of DNA, such as in bacterial typing for epi-
so that the DNA will make net progress in one demiological purposes. Enzymatic digestion of genomic
dimension. The parameters for this type of separa- DNA will yield a set of fragments that produce a band
tion must be matched to the DNA being separated pattern specific to each type of organism. By compar-
so that both large and small fragments have time ing band patterns, the similarity of organisms isolated
to reorient properly. For example, if timing is not from various sources can be assessed. This information
sufficient for reorientation of the large fragments, is especially useful in determining the epidemiology of
small fragments will preferentially reorient and infectious diseases, for example, identifying whether
move backward and gradually lose distance with two biochemically identical isolates have a common
respect to the large molecules, which will continue source.10
forward progress on the next pulse cycle. Unlike
PFGE that requires special equipment, FIGE can
be performed in a regular gel apparatus; however, Polyacrylamide Gels
its upper resolution limit is 2 megabases compared Very small DNA fragments, single-stranded DNA, RNA,
with 5 megabases for PFGE. and proteins are best resolved on polyacrylamide gels
in polyacrylamide gel electrophoresis (PAGE). Acryl-
amide, in combination with the cross-linker methylene
The large molecules resolved by these methods must be bisacrylamide (Fig. 4.5), polymerizes into a matrix that
protected from breakage and shearing. Therefore, spec- has consistent resolution characteristics (Fig. 4.6).
imens for PFGE analysis are immobilized in an agarose Unlike agarose, which is a natural polymer from
plug before cell lysis. Further purification and enzymatic living organisms, polyacrylamide is a synthetic material.
digestion of the DNA are also performed while the DNA This allows precise control of the polymer properties
is immobilized in the agarose plug. After treatment, and higher resolution than can be achieved with agarose.
CH2 CH CH2 CH CH2 CH CH2 CH CH2 CH

Persulfate
C O + C O C O C O C O
TEMED
NH2 NH NH2 NH NH2
FIGURE 4.5 The repeating unit Acrylamide CH2 CH2
of polyacrylamide is acrylamide;
NH NH2 NH NH2
bis introduces branches into the
polymer. In addition to concentra- C O C O C O C O
tion, the acrylamide:bis ratio will
determine the degree of branching CH2 CH CH2 CH CH2 CH CH2 CH
H
and the sieving properties of the bis
polymer. Polyacrylamide
The composition of polyacrylamide gels is represented

as the total percentage concentration (w/v) of monomer
(acrylamide with cross-linker), T, and the percentage of
monomer that is cross-linker, C. For example, a 6% 19:1
acrylamide:bis gel has a T value of 6% and a C value of
1/20, or 5%.
Unlike agarose gels that polymerize upon cooling,
polyacrylamide gels require a catalyst. The catalyst may
be the nucleating agents ammonium persulfate (APS) plus
800 bp
N,N,N′,N′-tetramethylethylenediamine (TEMED) or light
activation. APS produces free oxygen radicals in the pres-
ence of TEMED to drive the polymerization mechanism.
500 bp Alternatively, free radicals are generated by a photo-
chemical process using riboflavin plus TEMED. Excess
oxygen inhibits the polymerization process. Therefore,
de-aeration, or the removal of air, of the gel solution is
done before the addition of the nucleating agents.
Polyacrylamide gels for nucleic acid separation can
be very thin, for example, 50 μm, making gel prepara-
200 bp tion difficult. Available systems are designed to facili-
tate the preparation of single and multiple gels. A more
convenient, albeit more expensive, alternative is to use
FIGURE 4.6 Resolution of double-stranded DNA fragments preformed polyacrylamide gels to avoid the hazards of
on a 5%, 19:1 acrylamide:bis gel.
working with acrylamide and the labor time involved in
With single-base resolution, polyacrylamide gels are used gel preparation. Use of preformed gels must be sched-
for nucleic acid sequencing, mutation analyses, nuclease uled, keeping in mind the limited shelf life of the product.
protection assays, and other applications requiring high The main advantage of polyacrylamide over agarose
resolution of nucleic acids. Acrylamide is supplied to is the higher resolution capability of polyacrylamide for
the laboratory in several forms. The powdered form is small fragments. A variation of 1 bp in a 1-kb molecule
a dangerous neurotoxin and must be handled with care. (0.1% difference) can be detected in a polyacrylamide
Commercial solutions of mixtures of acrylamide and gel. Another advantage of polyacrylamide is that there
bis-acrylamide are less hazardous and more convenient is not as much difference in batches obtained from dif-
to use. Preformed gels are the most convenient because ferent sources as there is in agarose. Further, altering T
the procedure for preparation of acrylamide gels is more and C in a polyacrylamide gel can change the pore size,
difficult than that for agarose gels. and therefore the sieving properties, in a predictable and
reproducible manner. Increasing T decreases the pore
size proportionally. The minimum pore size (highest res-
Advanced Concepts olution for small molecules) occurs at a C value of 5%.
Variation of C above or below 5% will increase pore
Different cross-linkers affect the physical nature size. Usually, C is set at 3.3% (29:1) for native and 5%
of the acrylamide mesh. Piperazine diacrylate can (19:1) for standard DNA and RNA gels (see Table 4.2).
reduce the background staining that may occur
when the gel is stained. N,N ′-bisacrylylcystamine
and N,N ′,z-diallyltartardiamide enable gels to be CAPILLARY ELECTROPHORESIS
solubilized to facilitate extraction of separated
products. The widest application of capillary electrophoresis has
been in the separation of organic chemicals such as
TABLE 4.2 Choice of Polyacrylamide + –

–
Concentration for DNA Gels*14 + –
+ + + –
–
Separation Range
Acrylamide Concentration (%) (size in bp)
Net flow
3.5 100–1,000
5.0 80–500 Laser
8.0 60–400 +
–
12.0 40–200 Net flow
20.0 10–100 Detector
*The indicated figures are referring to gels run in TBE buffer. Voltages over
8 V/cm may affect these values.
pharmaceuticals and carbohydrates. It has also been Buffer Buffer

applied to the separation of inorganic anions and metal
ions. For these applications, capillary electrophoresis High voltage
has the advantages of faster analytical runs and lower
FIGURE 4.7 Capillary electrophoresis separates particles by
cost per run than other separation methods. Capillary
size (small, fast migration; large, slow migration) and charge
electrophoresis is also used for the separation and anal- (negative, fast migration; positive, slow migration). Because
ysis of nucleic acids. the size and charge of DNA work counter to each other, a
In this type of electrophoresis, the analyte is resolved polymer (gel) in the capillary will resolve the DNA fragments
in a thin glass (fused silica) capillary that is 30 to 100 cm mostly according to size.
in length with an internal diameter of 25 to 100 μm.
Fused silica is used as the capillary tube because it is
the most transparent material allowing for the passage Nucleic acids do not separate well in solution. As the
of fluorescent light. The fused silica is covered with a size or length of a nucleic acid increases (slowing migra-
polyimide coating for protection, except for an uncoated tion), so does its negative charge (speeding migration),
window where laser light excites the fluorescent mol- effectively confounding the charge/mass resolution.
ecules attached to the fragments as they pass a detec- Introducing a polymer inside the capillary establishes
tion device. The capillary has a negative charge along resolution by impeding nucleic acid migration according
its walls, generated by the dissociation of hydroxyl to size more than charge. It is important that the nucleic
ions from the molecules of silicone. This establishes an acid be completely denatured (single stranded) so that it
electro-osmotic flow when a current is introduced along will be separated according to its size because the sec-
the length of the capillary. Under the force of the current, ondary structure will affect the migration speed.
small and negatively charged molecules migrate faster Fluorescent labels are covalently attached to nucleic
than large and positively charged molecules (Fig. 4.7). acids to be separated by capillary electrophoresis. This
Optimal separation requires the use of the proper buffer can be done enzymatically or by polymerase chain
to ensure that the solute being separated is charged. reaction priming of DNA synthesis across the region of
Capillary electrophoresis was originally applied to interest with single-stranded DNA primers, one of which
molecules in solution. Separation was based on their carries a fluorescent molecule at the 5′ end. These pro-
size and charge (charge/mass ratio). Negatively charged cesses are described in Chapter 6, “Nucleic Acid Ampli-
molecules are completely ionized at high pH, whereas fication,” and Chapter 9, “DNA Sequencing.” Generally,
positively charged solutes are completely protonated in picogram to nanogram quantities of fluorescently labeled,
low-pH buffers. denatured nucleic acid in buffer containing formamide
are introduced to the capillary, which is held at a dena- accomplished through the electrochemical characteris-
turing temperature, usually 50°C to 60°C. tics of the buffer components.
The sample goes into the capillary through electroki- A buffer is a solution of a weak acid and its conju-
netic, hydrostatic, or pneumatic injection. Electroki- gate base. The pH of a buffered solution remains con-
netic injection is used for nucleic acid analysis. In this stant as the buffer molecules take up or release protons
process, a platinum electrode close to the end of the cap- given off or absorbed by other solutes. The equilibrium
illary undergoes a transient high-positive charge to draw between acid and base in a buffer is expressed as the
the sample to the end of the capillary. When the current dissociation constant, Ka:
is established with a positive charge on the opposite [ H + ][ A − ]
end of the capillary, the nucleic acids migrate into and Ka =
[ HA ]
through the capillary. Capillary electrophoresis is anal-
ogous to gel electrophoresis with regard to the electro- where [H+], [A−], and [HA] represent the dissociated
phoretic parameters for the resolution of nucleic acids. proton, conjugate base, and nondissociated acid concen-
The capillary’s small volume, as compared with that trations, respectively. Ka is most commonly expressed as
of a slab gel, can dissipate heat more efficiently during its negative logarithm, pKa, such that
the electrophoresis process. More efficient heat dissipa- pK a = − log K a
tion allows running the samples at a higher charge per A pKa of 2 (Ka = 10−2) favors the release of protons. A
unit area, which means that the samples migrate faster, pKa of 12 (Ka = 10−12) favors the association of protons.
thereby decreasing the resolution (run) time. A given buffer maintains the pH of a solution near
Nucleic acid resolution by capillary electrophoresis is its pKa. The amount the pH of a buffer will differ from
used extensively in forensic applications and parentage the pKa is expressed as the Henderson–Hasselbalch
testing performed by analyzing short DNA fragments. It equation:
has other applications in the clinical laboratory, such as
[ basic form]
clonality testing, microsatellite instability detection, and pH = pK a + log
bone marrow engraftment analysis. Specially designed [acidic form]
software can use differentially labeled molecular-weight If the acidic and basic forms of the buffer in solution are
markers or allelic markers that, when run through the of equal concentration, pH = pKa. If the acidic form pre-
capillary with the sample, help to identify sample bands. dominates, the pH will be less than the pKa; if the basic
Compared with traditional slab gel electrophoresis, form predominates, the pH will be greater than the pKa.
the capillary system has the advantages of increased sen- The Henderson–Hasselbalch equation predicts that in
sitivity and immediate detection. With multiple color- order to change the pH of a buffered solution by one
detection systems, standards, controls, and test samples point, either the acidic or basic form of the buffer must
are run through the capillary together, thereby eliminat- be brought to a concentration of 1/10 that of the other
ing the lane-to-lane variations that can occur across a form. Therefore, the addition of acid or base will barely
gel. Although instrumentation for capillary electropho- affect the pH of a buffered solution as long as the acidic
resis is costly and detection requires fluorescent labeling or basic forms of the buffer are not depleted.
of samples, labor and runtime are greatly decreased as Control of the pH of a gel by the buffer protects
compared with gel electrophoresis. In addition, analyti- sample molecules from damage. Furthermore, the
cal software programs automatically analyze the results current through the gel is carried by buffer ions, pre-
that are gathered by the detector in the capillary electro- venting severe fluctuations in the pH of the gel. A buffer
phoresis instrument. concentration must be high enough to provide sufficient
acidic and basic forms to buffer its solution. Raising the
buffer concentration, however, also increases the con-
BUFFER SYSTEMS ductivity of the electrophoresis system, generating more
heat at a given voltage. This can lessen gel stability and
The purpose of a buffer system is to carry the current increase sample denaturation. High buffer concentra-
and protect the samples during electrophoresis. This is tions must therefore be offset by low voltage.
In order for nucleic acids to migrate properly, the gel 0.04 M Tris-base, 0.005 M sodium acetate, 0.002 M
must be immersed in a buffer that conducts the electric EDTA), are most commonly used for electrophoresis
current efficiently in relation to the buffering capacity. of DNA. TBE has a greater buffering capacity than
Ions with high-charge differences, +2, –2, +3, and so on, TAE. Although the ion species in TAE are more easily
move through the gel more quickly; that is, they increase exhausted during extended or high-voltage electrophore-
conductivity without increasing buffering capacity. This sis, DNA will migrate twice as fast in TAE than in TBE
results in too much current passing through the gel as well in a constant current. When using any buffer, especially
as faster depletion of the buffer. Therefore, buffer com- TBE and TPE, the gel can overheat when run at high
ponents such as Tris base [2-amino-2-(hydroxymethyl)- voltage in a closed container. Finally, stock solutions of
1,3-propanediol] or borate are preferred because they TBE are prone to precipitation. This can result in dif-
remain partly uncharged at the desired pH and thus ferences in concentration between the buffer in the gel
maintain constant pH without high conductivity. In addi- and the running buffer. Such a gradient will cause local-
tion to pKa, charge, and size, other buffer characteristics ized distortions in nucleic acid migration patterns, often
that might be taken into account when choosing a buffer causing a salt wave that is visible as a sharp horizontal
include toxicity, interaction with other components, sol- band through the gel.
ubility, and ultraviolet (UV) absorption.
Buffer Additives
Advanced Concepts Buffer additives modify sample molecules in ways that
affect their migration. Examples of these additives are
The Henderson–Hasselbalch equation also pre- formamide, urea, and detergents. With regard to nucleic
dicts the concentration of the acidic or basic forms acids, denaturing agents, such as formamide or urea,
at a given pH. It can be used to calculate the state break hydrogen bonds between complementary strands
of ionization of a species in solution, that is, the or within the same strand of DNA or RNA. The confor-
predominance of acidic or basic forms. A buffer mation or solubility of molecules can be standardized
should be chosen that has a pKa within a half point by the addition of one or both of these agents. Forma-
of the desired pH, which is about 8.0 for nucleic mide and heat added to DNA and RNA break and block
acids. the hydrogen-bonding sites, hindering complementary
sequences from reannealing. As a result, the molecules
become long, straight, unpaired chains. Urea and heat
in the gel systems maintain this conformation such that
Advanced Concepts intrachain hybridization (folding) of the nucleic acid
molecules does not affect migration speeds, and sepa-
The migration of buffer ions is not restricted by ration occurs strictly according to the size or length of
the gel matrix, so the speed of their movement the molecule.
under a current is governed strictly by the size of Electrophoresis of RNA requires different condi-
the ion and its charge (charge/mass ratio). Tris is tions imparted by different additives from those that
a relatively large molecule, with a low charge-to- are used with DNA. Because RNA is single stranded
mass ratio, and it moves through the current rela- and longer molecules tend to fold to optimize internal
tively slowly, even at high concentrations, giving hydrogen bonding, they must be completely denatured
increased buffering capacity. to prevent folding in order to accurately determine the
size by migration in a gel system. The secondary struc-
tures formed in RNA are strong and more difficult to
The Tris buffers, Tris borate EDTA (TBE; 0.089 M Tris- denature than DNA homologies. Denaturants used for
base, 0.089 M boric acid, 0.0020 M EDTA), Tris phos- RNA include methylmercuric hydroxide (MMH), which
phate EDTA (TPE; 0.089 M Tris-base, 1.3% phosphoric reacts with amino groups on the RNA to prevent base
acid, 0.0020 M EDTA), and Tris acetate EDTA (TAE; pairing between complementary nucleotides and with
+ Buffer solution Gel Electrode

–
Gel
Buffer
Magnet
Magnetic stirrer
Tubing Tubing
Peristaltic – +
pump
(Black) (Red)
FIGURE 4.8 A peristaltic pump is used to recirculate buffer
from the cathode to the anode end while running a denaturing
FIGURE 4.9 A typical horizontal submarine gel system. A
red connector is attached to the positive outlet on the power
gel.
supply, and a black connector is attached to the negative port.
Nucleic acid will migrate to the positive (red) pole.
aldehydes (e.g., formaldehyde, glyoxal), which also
disrupt base pairing. MMH is not used routinely because and makes a continuous system through which the
of its extreme toxicity. current flows. The thickness of the gel and the volume
Examples of RNA electrophoresis buffers are 10-mM of the buffer affect the current, and therefore the migra-
sodium phosphate, pH 7, and MOPS buffer [20 mM tion of the sample, so these parameters are kept constant
3-(N-morpholino) propanesulfonic acid, pH 7, 8 mM for consistent results. Because the gel is submerged
sodium acetate, 1 mM EDTA, pH 8]. The RNA sample throughout the loading and electrophoresis process, hori-
is incubated in dimethyl sulfoxide, 1.1 M glyoxal zontal gels are sometimes referred to as submarine gels.
(ethane 1.2 dione) and 0.01 M sodium phosphate, pH Once the sample is introduced into the gel, the elec-
7, to denature the RNA prior to loading the sample on trodes are connected to the power source. The power
the gel. Due to pH drift (decrease of pH at the cathode supply will deliver voltage, setting up a current that
[–] and an increase of the pH at the anode [+]) during will run through the gel buffer and the gel, carrying
the run, the buffer may have to be recirculated from the the charged sample through the gel matrix at a speed
anode end of the bath to the cathode end (Fig. 4.8). This corresponding to the charge/mass ratio of the sample
is accomplished by using a peristaltic pump or stopping molecules.
the gel at intervals and transferring the buffer from the Horizontal agarose gels are cast as square or rectan-
cathode to the anode ends. gular slabs of varying size. Commercial gel boxes come
with casting trays that mold the gel to the appropriate
size for the gel box. The volume of the gel solution will
ELECTROPHORESIS EQUIPMENT determine the thickness of the gel. Agarose, supplied
as a dry powder, is mixed at a certain percentage (w/v)
Gel electrophoresis is performed in one of two confor- with electrophoresis buffer and heated on a heat block
mations, horizontal or vertical. In general, agarose gels or by microwave to dissolve and melt the agarose. The
are run horizontally, and polyacrylamide gels are run molten agarose is cooled to between 55°C and 65°C, and
vertically. This, however, is not always the case. the appropriate volume is poured into the casting tray
Horizontal gels are run in acrylic gel boxes or baths as dictated by the gel box manufacturer or application.
that are divided into two parts, with a platform in the A comb is then inserted into the top of the gel to create
middle on which the gel rests (Fig. 4.9). The electrodes holes, or wells, in the gel into which the sample will be
(platinum wires) placed across the box at each end of loaded. The size of the teeth in the comb will determine
the bath compartments are connected to a power supply the capacity of the well for the sample, and the number
through the walls of the container. The gel is submerged of teeth in the comb will determine the number of wells
in electrophoresis buffer that fills both compartments that are available in the gel to receive the samples. The
gel is then allowed to cool, during which time it will

solidify. After the gel has polymerized, the comb is care-
fully removed, and the gel is placed into the gel box and
submerged in the electrophoresis buffer.
There is a significant shock hazard if contact is made
with the gel buffer while the current is on. For safety pur-
poses, gel baths are designed to stop the current when a
protective cover is removed from the gel bath. Enclosed FIGURE 4.10 Combs for polyacrylamide electrophoresis.
cassettes containing gel, buffer, electrodes, and detection Regular combs (top) have teeth that form the wells in the gel.
do not require submersion in running buffer. The sample Sharkstooth combs (bottom) are placed onto the polymerized
is loaded directly into the wells of these systems. gel, and the sample is loaded between the teeth of the comb.
Vertical gels are cast between glass plates that are
separated by spacers. The spacers determine the thick- Vertical gel boxes have separate chambers connected
ness of the gel, ranging from 0.05 to 4 mm. The bottom by the gel itself. Electrodes are attached to the upper and
of the gel is secured by tape or by a gasket in specially lower buffer chambers to set up the current that will run
designed gel casting trays. After the addition of catalyst through the gel.
and nucleating agents, the liquid acrylamide is poured or The gel must be in place before filling the upper
forced between the glass plates with a pipet or a syringe. chamber with buffer. Some systems have a conductive
The comb is then placed on the top of the gel. For plate attached to the back of the gel to maintain a con-
light-activated polymerization, the gel between the glass stant temperature across the gel. Maintaining constant
plates is exposed to a light source. During this process, temperature is more of a problem with vertical gels
it is important to prevent air from getting into the gel or because the outer edges of the gel cool more than the
beneath the comb. Bubbles will form discontinuities in center, slowing migration in the outer lanes compared
the gel, and oxygen will inhibit the polymerization of with lanes in the center of the gel. This is called “gel
the acrylamide. The comb is of a thickness equal to that smiling” because similar-sized bands in the cooler outer
of the spacers so that the gel will be the same thickness lanes will migrate slower than comparable bands in the
throughout. inside lanes. Vertical gel systems can range from large
As with horizontal gels, the number and size of sequencing gels (35 × 26 cm) to minigels (8 × 10 cm).
the comb’s teeth determine the number of wells in the Some minigel systems accommodate two or more gels
gel and the sample volume that can be added to each at a time (Fig. 4.11). Minisystems are used extensively
well. Specialized combs, called sharkstooth combs, for analyses that do not require single base-pair resolu-
facilitate lane to lane band comparisons (Fig. 4.10). tion. The larger systems have been used for procedures
These combs are placed upside down (teeth up, not in requiring higher resolution but are being replaced by
contact with the gel) to form a straight surface on the capillary electrophoresis for many applications.
gel during polymerization. After polymerization is com- Vertical gels are loaded from the top, below a layer of
plete, the comb is removed and placed tooth-side down buffer in the upper chamber. Long, narrow, gel-loading
on top of the gel for loading. With this configuration, pipette tips that deposit the sample neatly on the floor of
the spaces between the comb teeth form the well walls. the well increase band resolution and sample recovery.
The advantage to this arrangement is that the result-
ing gel lanes are immediately adjacent to one another.
When used, standard combs are removed before the Advanced Concepts
gel is loaded, whereas the samples are loaded while the
sharkstooth comb is in place. When the standard combs Self-contained agarose gel systems have been
are removed from the gel, care must be taken not to developed to facilitate the electrophoresis process.
break or displace the “ears” that were formed by the They are manufactured in closed plastic cas-
spaces between the teeth in the comb that separate the settes containing buffer, gel, and stain. These are
gel wells.
+
–
Buffer
+
Gel
plates
Electrode
Gel
Buffer
FIGURE 4.11 A typical vertical gel apparatus. Polymerized gels are clamped into the gel insert and placed in the gel bath. The
positive electrode will be in contact with the bottom of the gel and the buffer, filling about a third of the gel bath. The negative
electrode will be in contact with the top of the gel and a separate buffer compartment in the top of the insert.
the molecules separated in one direction in the tube are

convenient for routine use, but they restrict the gel then further separated in a second dimension through the
configuration, that is, the number and size of wells, slab gel. Two-dimensional gel electrophoresis is mostly
and so forth. Also, the percentage of agarose or applied to protein separations.
acrylamide is limited to what is available from the
manufacturer. Furthermore, the separated nucleic Gel Loading
acids cannot easily be removed from these closed
cassettes, limiting post-electrophoretic procedures. Prior to loading the sample containing isolated nucleic
acid onto a gel, tracking dye and a density agent are
added to the sample. The density agent (Ficoll, sucrose,
or glycerol) increases the density of the sample as com-
Polyacrylamide gels can also be cast in tubes for isoelec- pared with the electrophoresis buffer. When the sample
tric focusing or two-dimensional gel electrophoresis. solution is dispensed into the wells of the gel below the
The tubes containing the gels are placed in a chamber surface of the buffer, it sinks into the well instead of
separated like those for vertical slab gels. The tubes diffusing in the buffer. Although samples are pipetted
are held in place by gaskets in the upper chamber. into the wells as close as possible to the well floor, the
When a tubular gel is placed at the top of a slab gel, density agent mixed with the sample allows loading from
TABLE 4.3 Tracking Dye Comigration* Advanced Concepts

Bromophenol Blue Xylene Cyanol A type of “bufferless” electrophoresis system sup-
Gel % (Nucleotides) (Nucleotides) plies buffer in gel form or strips. These are laid next
Agarose to the preformed gel on a platform that replaces
the electrophoresis chamber. These systems can
0.5–1.5 300–500 4,000–5,000 offer the additional advantage of precise tempera-
2.0–3.0 80–120 700–800
ture control during the run.
4.0–5.0 20–30 100–200
PAGE
4 95 450
Advanced Concepts
6 60 240 Gels in cassette systems and gel strip systems can
be loaded without loading buffer because the wells
8 45 160
are “dry,” precluding the need for density gradi-
10 35 120 ents. These systems also have automatic shut-off
at the end of the run, so tracking dye is usually not
12 20 70 necessary, although some systems have a tracking
20 12 45 dye built into the gel and/or buffer.
*Migration depends on buffer type (TAE, TBE, or TPE) and the formulation of
agarose, acrylamide, and bis.
DETECTION SYSTEMS
far enough away so as not to risk damaging the well. If a
The agents used most frequently for visualization of
well is damaged, the tracking dye in the sample will be
bands after electrophoresis are fluorescent dyes and
seen seeping out of the well and under the gel.
silver stain. Both of these types of dyes specifically
The tracking dyes are used to monitor the progress
associate with nucleic acid.
of the electrophoresis run. The dyes migrate at specific
speeds in a given gel concentration and usually run
ahead of the smallest fragments of DNA (Table 4.3). Fluorescent Dyes
Bromophenol blue is a tracking dye that is used for
Intercalating Agents
many applications, and xylene cyanol green is another
of the chromophores used as tracking dyes for both Intercalating agents intercalate, or stack, between the
agarose and polyacrylamide gels. Tracking dyes are not nitrogen bases in double-stranded nucleic acid. Ethid-
associated with the sample DNA, and thus they do not ium bromide (3,8-diamino-5-ethyl-6-phenylphenan-
affect the separation. The movement of the tracking dye thridinium bromide [EtBr]) is one of these agents and
is monitored, and when the dye approaches the end of was the most widely used dye in early DNA and RNA
the gel, or the desired distance, electrophoresis is termi- analyses. Because EtBr is carcinogenic, precautions
nated. Running a gel too long will result in loss of some are required to limit exposure. Under excitation with
or all of the desired bands because they will run off the UV light at 300 nm, EtBr in DNA emits visible light
bottom of the gel into the gel bath. Timers or automatic at 590 nm. Therefore, DNA separated in agarose or
power cutoffs are used to avoid loss of sample. Some gel acrylamide and exposed to EtBr will emit orange light
systems will automatically stop at the appropriate time when illuminated at 300 nm. After electrophoresis, the
for the gel size and current strength. agarose or acrylamide gel is soaked in a solution of
0.1- to 1-mg/mL EtBr in running buffer (TAE, TBE, or SYBR gold stain is a cyanine dye of proprietary
TPE) or in TE. Alternatively, dye is added directly to structure. Its fluorescence increases more than 1,000-
the gel before polymerization or to the running buffer. fold upon binding to double- or single-stranded DNA or
The latter two measures save time and allow visualiza- to RNA. Like SYBR green, SYBR gold is excited by
tion of the DNA during and immediately after the run; UV light (300-nm wavelength). SYBR gold emits light
however, dye added to the gel may form a bright front at 537 nm.
across the gel that could mask informative bands. Dye Although SYBR dyes have some advantages over
added to the running buffer produces consistent staining, EtBr, many laboratories continue to use the EtBr due to
although greater volumes of hazardous waste are gener- the requirement for special optical filters for detection of
ated by this method. To increase safety, noncarcinogenic SYBR green emission at 520- to 550-nm wavelengths
dyes with similar excitation and emission wavelengths and SYBR gold at 537 nm. Scanning and photographic
as EtBr have been developed. Alternatively, enclosed equipment optimized for EtBr would have to be mod-
gel systems contain EtBr inside a plastic-enclosed gel ified for optimal detection of the SYBR stains. New
cassette, limiting exposure and limiting waste. After instrumentation with more flexible detection systems
soaking or running in dye, the DNA illuminated with UV allows utilization of the SYBR and other fluorescent
light will appear as orange bands in the gel. The image stains. SYBR green is the preferred dye for real-time
is captured with appropriate filters by digital transfer to PCR methods.
analytical software.
Silver Stain
Minor Groove–Binding Dyes
Another sensitive staining system originally developed
SYBR green (N′,N′-dimethyl-N-[4-[(E)-(3-methyl-1,3- for protein visualization is silver stain. After electropho-
benzothiazol-2-ylidene)methyl]-1-phenylquinolin- resis, the sample is fixed with methanol and acetic acid.
1-ium-2-yl]-N-propylpropane-1,3-diamine) is one of a The gel is then impregnated with ammoniacal silver
set of stains introduced in 1995 as another type of nucleic (silver diamine) solutions or silver nitrate in a weakly
acid–specific dye system. The related dyes include acid solution.13 Interaction of silver ions with acidic or
SYBR green I used for double-stranded DNA staining; nucleophilic groups on the target results in crystalliza-
SYBR green II, for single-stranded DNA or RNA stain- tion or deposition of metallic silver under optimal pH
ing; and SYBR gold, for both DNA and RNA staining. conditions. The insoluble black silver salt precipitates
SYBR green I differs from EtBr in that it does not inter- upon introduction of formaldehyde in a weak acid solu-
calate between bases; it sits in the minor groove of the tion, or alkaline solution for silver nitrate. Of the two
double helix. SYBR green in association with DNA or procedures, silver diamine is best for thick gels, whereas
RNA also emits light in the orange range (522 nm). In silver nitrate is considered to be more stable.14 Optimi-
agarose gel electrophoresis, SYBR staining is 25 to 100 zation of silver staining has continued to simplify and
times more sensitive than EtBr (detection level: 60 pg increase its efficiency. One method reports sensitivity of
of double-stranded DNA versus 5 ng for EtBr). This is detection as low as 14.6 pg in 6 to 7 minutes using only
due, in part, to background fluorescence from EtBr in three reagents (silver nitrate, sodium hydroxide, and
agarose. A 1× dilution of the manufacturer ’s 10,000× formaldehyde).15
stock solution of SYBR green I in TAE, TBE, or TE is Silver staining avoids the hazards of the interca-
used in the methods described for EtBr. SYBR green can lators, but silver nitrate is also a biohazard and must
also be added directly to the DNA sample before electro- be handled accordingly. In addition, silver staining
phoresis. DNA prestaining decreases the amount of dye is more complicated than simple fluorescent stains.
required for DNA visualization but lowers the sensitiv- Color development must be carefully watched in
ity of detection and may, at higher DNA concentrations, order to stop the reaction once the optimal signal is
interfere with DNA migration through the gel.11 Because reached. Overdevelopment of the color reaction will
SYBR green is not an intercalating agent, it is not as result in high backgrounds and masking of results. The
mutagenic and is therefore safer to use.12 increased sensitivity of this staining procedure, however,
makes up for its limitations. It is especially useful for 9. Name two dyes that are used to monitor the
protein analysis and for detection of limiting amounts migration of nucleic acid during electrophoresis.
of product.
10. When a DNA fragment is resolved by slab gel
electrophoresis, a single sharp band is obtained.
What would the equivalent observation be if
STUDY QUESTIONS this fragment had been fluorescently labeled and
resolved by capillary electrophoresis?
1. You wish to perform an electrophoretic resolution
of your restriction enzyme–digested DNA. The References
sizes of the expected fragments range from
1. Tiselius A. A new apparatus for electrophoretic analysis of colloi-
100 to 500 bp. You discover two agarose gels dal mixtures. Transactions of the Faraday Society 1937;33:524.
polymerizing on the bench. One is 0.5% agarose; 2. Kunkel H, Slater RJ. Zone electrophoresis in a starch supporting
the other is 2% agarose. Which one might you use medium. Proceedings of the Society of Experimental Biology and
to resolve your fragments? Medicine 1952;80:42–44.
3. Smithies O, Walker NF. Genetic control of some serum proteins in
normal humans. Nature 1955;176:1265–1266.
2. After completion of the electrophoresis of DNA 4. Smithies O, Poulik MD. Two-dimensional electrophoresis of
fragments along with the proper molecular-weight serum proteins. Nature 1956;177:1033.
standard on an agarose gel, suppose the outcomes 5. Carle G, Frank M, Olson MV. Electrophoretic separation of large
in (a) and (b) were observed. What might be the DNA molecules by periodic inversion of the electric field. Science
1986;232:65–68.
explanations for each outcome?
6. Chu G, Vollrath D, Davis RW. Separation of large DNA mol-
a. The gel is blank (no bands, no molecular-weight ecules by contour-clamped homogeneous electric fields. Science
1986;234:1582–1585.
standard).
7. Gardiner K, Laas W, Patterson DS. Fractionation of large mamma-
b. Only the molecular weight standard is visible. lian DNA restriction fragments using vertical pulsed-field gradient
gel electrophoresis. Somatic Cell and Molecular Genetics 1986;12:
3. How does PFGE separate larger fragments more 185–195.
efficiently than standard electrophoresis? 8. Southern E, Anand R, Brown WRA, Fletcher DS. A model for the
separation of large DNA molecules by crossed field gel electro-
phoresis. Nucleic Acids Research 1987;15:5925–5943.
4. A 6% solution of 19:1 acrylamide:bis-acrylamide is 9. Gemmill R. Pulsed field gel electrophoresis. In Advances of Elec-
mixed, de-aerated, and poured between glass plates trophoresis. Edited by Chrambach A, Dunn MJ, Radola, BJ. Wein-
for gel formation. After an hour, the solution is still heim, Germany: VCH, 1991. pp. 1–48.
liquid. What might be one explanation for the gel 10. Donnenberg M, Narayanan S. How to diagnose a foodborne
illness. Infectious Disease Clinics of North America 2013;27:3.
not polymerizing?
11. Miller S, Taillon-Miller P, Kwok P. Cost-effective staining of
DNA with SYBR green in preparative agarose gel electrophoresis.
5. A gel separation of RNA yields aberrantly BioTechniques 1999;27:34–36.
migrating bands and smears. Suggest two possible 12. Singer V, Lawlor, TE, Yue, S. Comparison of SYBR green I
explanations for this observation. nucleic acid gel stain mutagenicity and ethidium bromide muta-
genicity in the Salmonella/mammalian microsome reverse muta-
tion assay (Ames test). Mutation Research 1999;439:37–47.
6. Why does DNA not resolve well in solution 13. Rabilloud T. A comparison between low background silver
(without a gel matrix)? diamine and silver nitrate protein stains. Electrophoresis 1992;13:
429–439.
7. Why is SYBR green less toxic than EtBr? 14. Merrill C. Gel-staining techniques. Methods in Enzymology
1990;182:477–488.
15. Liu W, Li R, Ayalew H, Xia Y, Bai G, Yan G, Siddique KH,
8. What are the general components of loading buffer Guo P. Development of a simple and effective silver stain-
used for introducing DNA samples to submarine ing protocol for detection of DNA fragments. Electrophoresis
gels? 2017;38(8):1175–1178.
Chapter 5
Analysis and Characterization
of Nucleic Acids and Proteins
Outline ARRAY-BASED HYBRIDIZATION

Dot/Slot Blots
RESTRICTION ENZYME MAPPING OF DNA Genomic Array Technology
CRISPR ENZYME SYSTEMS Macroarrays
HYBRIDIZATION TECHNOLOGIES Microarrays
Southern Blots Bead Array Technology
Restriction Enzyme Cutting and Resolution SOLUTION HYBRIDIZATION
Preparation of Resolved DNA for Blotting (Transfer)
Blotting (Transfer)
PROBE HYBRIDIZATION
Northern Blots Objectives
Western Blots
PROBES 5.1 Describe how restriction enzyme sites are mapped
DNA Probes on DNA.
RNA Probes 5.2 Construct a restriction enzyme map of a DNA
Other Nucleic Acid Probe Types plasmid or fragment.
Protein Probes 5.3 Diagram the Southern blot procedure.
Probe Labeling 5.4 Explain depurination and denaturation of resolved
Nucleic Acid Probe Design DNA.
HYBRIDIZATION CONDITIONS, STRINGENCY 5.5 Describe the procedure involved in blotting
DETECTION SYSTEMS (transfer) DNA from a gel to a membrane.
INTERPRETATION OF RESULTS 5.6 Discuss the purpose and structure of probes that
are used for blotting procedures.
5.7 Define hybridization, stringency, and melting
temperature.
5.8 Calculate the melting temperature of a given
sequence of double-stranded DNA.
112
Chapter 5 • Analysis and Characterization of Nucleic Acids and Proteins 113
5.9 Compare and contrast radioactive and with the enzyme, the resulting fragments are separated
nonradioactive DNA detection methods. by gel electrophoresis. The gel image reveals four
5.10 Compare and contrast dot and slot blotting fragments, labeled A, B, C, and D, produced by PstI
methods. (Fig. 5.1). From the number of fragments, one can
5.11 Describe microarray methodology. deduce the number of PstI sites: three. The sizes of the
5.12 Discuss solution hybridization. fragments, as determined by comparison with known
molecular-weight standards, indicate the distance
between PstI sites or from one PstI site to the end of the
fragment. Although PstI analysis of this fragment yields
There is a growing assortment of methods for a characteristic four-band restriction pattern, it does not
analysis and characterization of nucleic acids. Advances indicate the order of the four restriction products in the
in DNA sequencing technology have made analysis at original fragment.
single-nucleotide resolution relatively straightforward. To begin to determine the order of the restriction
Sequencing technology is described in Chapter 9, “DNA fragments, another enzyme is used, for example, BamHI.
Sequencing.” Even so, some tests require detection of Cutting the same fragment with BamHI yields two
known sequence variations at a single codon or even a pieces, indicating one BamHI site in this linear fragment
single nucleotide, for which the effort and expense of (see Fig. 5.1). In this figure, observe that one restriction
sequencing are less efficient. In these cases, methods product (F) is much larger than the other (E). This means
derived even before the development of current sequenc- that the BamHI site is close to one end of the fragment.
ing technology are most appropriate. When the fragment is cut simultaneously with PstI and
BamHI, five products are produced, with PstI product
A cut into two pieces by BamHI. Because the BamH1
RESTRICTION ENZYME MAPPING OF DNA site is known to be close to one end of the fragment,
PstI fragment A is on one end of the DNA fragment. By
Clinical and forensic analyses require characterization of measuring the number and length of products produced
specific genes or genomic regions at the molecular level. by other enzymes, the restriction sites can be placed in
Because of their sequence-specific activity, restriction linear order along the DNA sequence. Figure 5.2 shows
endonucleases provide a convenient tool for molecular two possible maps based on the results of cutting the
characterization of DNA. fragment with PstI and BamHI. With adequate enzymes
Restriction enzymes commonly used in the laboratory and enzyme combinations, a detailed map of this frag-
(type II restriction enzymes) have 4 to 6 base-pair (bp) ment is generated.
recognition sites, or binding/cutting sites, on the DNA. Mapping of a circular plasmid is slightly different
Any 4- to 6-bp nucleotide sequence occurs at random because there are no free ends (Fig. 5.3). The example
in a sufficiently long stretch of DNA. Therefore, restric- shown in the figure is a 4-kb-pair circular plasmid with
tion sites will occur in all DNA molecules of sufficient one BamHI site and two XhoI sites. Cutting the plasmid
length. The location of restriction sites will differ among with BamHI will yield one fragment. The size of the
DNA molecules with different sequences. Restric- fragment is the size of the plasmid, 4 kb in this example.
tion site mapping (i.e., determining where in the DNA Two fragments released by XhoI indicate that there are
sequence a particular restriction enzyme recognition site two XhoI sites in the plasmid and that these sites are 1.2
is located) was developed using small circular bacterial and 2.8 kb pairs away from each other. As with linear
plasmids. The resultant maps were used to identify and mapping, cutting the plasmid with XhoI and BamHI at
characterize naturally occurring plasmids and to engi- the same time will start to order the sites with respect to
neer the construction of recombinant plasmids. one another on the plasmid. One possible arrangement
To make a restriction map, DNA is exposed to is shown in Figure 5.3. As more enzymes are used, the
several restriction enzymes separately and then in differ- map becomes more detailed.
ent combinations. Take, for example, a linear fragment Under the proper reaction conditions, restriction
of DNA digested with the enzyme PstI. After incubation enzymes are highly specific for their recognition and
DNA
PstI Pst I PstI
A B C D
DNA
BamHI
E F
Pst I
+
Uncut Pst I Uncut BamHI Uncut Pst I BamHI BamHI
F
A *
*
D
B
C
E *
FIGURE 5.1 Restriction mapping of a linear DNA fragment (top, green bar). The fragment is first cut with the enzyme PstI. Four
fragments result, as determined by agarose gel electrophoresis, indicating that there are three PstI sites in the linear fragment. The
size of the pieces indicates the distance between the restriction sites. A second cut with BamHI (bottom) yields two fragments,
indicating one site. Because one BamHI fragment (E) is very small, the BamHI site must be near one end of the fragment. Cutting
with both enzymes indicates that the BamHI site is in the PstI fragment A.
A B C D cutting properties. Under nonstandard conditions,

however, some restriction enzymes will bind to and cut
BamHI PstI Pst I Pst I sequences other than the expected recognition sequence.
This altered specificity is called star activity. The pro-
A C D B pensity for star activity varies among enzymes.1 Thus,
the nature and degree of star activity depend on the
BamHI PstI Pst I PstI enzyme and the reaction conditions. Reaction conditions
FIGURE 5.2 Two possible maps inferred from the observa- that induce star activity include suboptimal buffer, con-
tions described in Figure 5.1. The BamHI site positions frag- tamination with organic solvents (e.g., ethanol), high
ment A at one end (or the other) of the map. Determination of concentrations of glycerol, prolonged reaction time, high
the correct map requires information from additional enzyme concentration of enzymes, pH greater than 8.0, and diva-
cuts. lent cation imbalance.
The pattern of fragments produced by restriction
enzyme digestion of a DNA fragment or region can
be used to identify that DNA. Because of inherited
BamHI
+
BamHI XhoI XhoI
BamHI
4.3 kb 4.0 kb
3.7 kb 2.8 kb 1.1 kb
2.3 kb 1.7 kb
1.7 kb XhoI
FIGURE 5.3 Restriction mapping of a plasmid. 1.9 kb
After incubating plasmid DNA with restriction 1.4 kb 1.2 kb 1.2 kb 1.2 kb
enzymes, agarose gel electrophoresis banding pat- 1.3 kb
terns indicate the number of restriction sites and the 1.1 kb XhoI
0.7 kb
distance between them.
or somatic differences in the nucleotide sequences in repeats. These spacer sequences serve as adaptive immu-
human DNA, the number and location of restriction nity with memory of the invading DNA. The locus also
sites for a given restriction enzyme are not the same in encodes an endonuclease, CRISPR-associated protein
all individuals. The resulting differences in the size or (Cas).
number of restriction fragments are restriction frag- To fend off an invader, short RNA sequences tran-
ment length polymorphisms (RFLPs). In addition to scribed from the CRISPR spacer regions (CRISPR RNA or
their use in epidemiological studies to identify micro- crRNA) are processed from larger pre-crRNA transcripts.
organisms and plasmids, RFLPs were the basis of the The crRNA then forms a complex with a trans-activating
first molecular-based human identification and mapping CRISPR RNA (tracrRNA) and Cas enzyme (Fig. 5.4).
methods. RFLPs are also used for the clinical analysis This complex, led to its target by the crRNA homology
of structural changes in chromosomes associated with where it binds and cuts the invading DNA. Cas9 requires
disease (translocations, deletions, insertions). a specific protospacer adjacent motif (PAM) to cut the
DNA. The protospacer is the part of the crRNA sequence
that is complementary to the target sequences incorpo-
CRISPR ENZYME SYSTEMS rated into the bacterial genome. The PAM varies depend-
ing on the bacterial species of the Cas gene. The Cas9
DNA cut sites of restriction enzymes used for DNA nuclease from Streptococcus pyogenes recognizes a PAM
analysis are limited to sequences recognized by the sequence of NGG directly 3′ of the target sequence in the
enzyme proteins. Another type of restriction system target DNA, on the complementary strand. The PAM may
found in archaea, gram-negative bacteria, and gram- facilitate the formation of the RNA:DNA hybrid between
positive bacteria guides a common enzyme to specific the crRNA and the target DNA.
sites determined by RNA components. This flexible There are three types of CRISPR/Cas systems: type
system has proven to be useful for manipulation of both I and II that target double-stranded DNA and type III
DNA sequence and RNA expression. that targets single-stranded DNA and RNA. The crRNAs
Clustered regularly interspaced short palindromic from each CRISPR locus are specifically processed by
repeats (CRISPRs) are classes of repeated DNA Cas and Cse proteins associated with that locus.2 The
sequences found in prokaryotic and archaebacterial type II system from S. pyogenes that encodes Cas9 is the
genomes. They are repeated sequences interrupted by most well studied.
spacer sequences matching the genome regions of plas- CRISPR/Cas9 has been used extensively in research
mids or bacteriophages that had previously infected as an efficient system to alter DNA at specific locations
the bacterium. DNA from new invaders is incorporated in the genome. Synthetic crRNA and tracRNA can be
into the CRISPR locus within a series of short (~20 bp) designed to lead the Cas9 endonuclease to the site of
Invading DNA
Target sequence PAM
Bacterial DNA
tracDNA Cas9 Cas genes crRNA Target crRNA Target crRNA
tracRNA Primary transcript FIGURE 5.4 Clustered regularly interspaced

short palindromic repeats (CRISPR) is a protec-
tive system that uses the invading DNA sequences
to target itself. The CRISPR locus consists of reg-
ularly spaced short palindromic repeats. Foreign
DNA sequences are incorporated into the bacterial
genome at the CRISPR repeat loci, interspersed
crRNA with target (invader) DNA, tracRNA the gene, and
the CRISPR-associated (Cas) operon. CRISPR
loci are then transcribed and processed into
Invading DNA crRNA. crRNA and tracRNA combine with the
Cas enzyme to find homology with invading DNA
Cas9 adjacent to the PAM sequence, at which site the
enzyme will cut the invading DNA.
Histooricaal Higghlligghtts Advanced Concepts

Before CRISPR was described, targeted genome
Targeted genome editing is the modification of a
editing was performed in vitro with transcription
sequence of interest in living cells or organisms.
activator–like effector nucleases (TALENs) and
Edits of early-stage embryos or even zygotes
zinc finger nucleases (ZFNs).3 These engineered
modify the genome in all the cells of an organ-
nucleases contained a customized target-se-
ism. The first disease gene repair was performed
quence-specific DNA-binding domain fused to a
in mice where a gene mutation that caused lens
nuclease that would cut DNA at any sequence.
clouding (cataracts) in mice was targeted with a
The nucleases made double-strand breaks (DSBs)
dominant normal gene using the CRISPR/Cas9
into targeted DNA sites. Designing and engi-
system.6 Modification of autologous hematopoietic
neering the proteins, however, was technically
stem cells using CRISPR technology is a promis-
demanding and time-consuming, limiting their
ing approach to the treatment of blood disorders
use in many applications.
currently treated with bone marrow transplants.7
choice, providing the specificity of restriction enzymes HYBRIDIZATION TECHNOLOGIES

with the versatility of guiding cuts to desired sequence
sites. Once the DNA is cleaved, the cell will repair the Procedures performed in the clinical molecular labora-
break by homologous recombination with a synthetic tory are aimed at specific targets in genomic DNA. This
donor template providing any desired sequence changes. requires visualization or detection of a particular gene or
CRISPR RNA can also lead activators or repressors region of DNA in the backdrop of all other genes. There
instead of Cas9 to gene-promoter sites and affect gene are several ways to find a target region of DNA, some
transcription. Specific regions can be visualized by of which require cloning of the region of interest. Early
bringing reporter molecules, such as green fluorescent cloning methods were complex, requiring screening of
protein, in place of Cas9.4,5 thousands of plasmids into which DNA regions were
TABLE 5.1 Hybridization Technologies
Hybridization Method Target Probe Purpose
Southern blot DNA Nucleic acid Gene structure
Northern blot RNA Nucleic acid Transcript structure, processing, gene expression
Western blot Protein Protein Protein processing, gene expression
Southwestern blot Protein DNA DNA-binding proteins, gene regulation
Eastern blot Protein Protein Modification of western blot using enzymatic detection (PathHunter);
also, detection of specific agriculturally important proteins28
Far-eastern blot29,30 Lipids (None) Transfer of high-performance liquid chromatography (HPLC)-

separated lipids to polyvinyl difluoride (PVDF) membranes for analysis
by mass spectrometry
randomly inserted. The first method for molecular anal- modifications of the procedure in order to analyze RNA
ysis of specific DNA sites within a complex background and protein.
without cloning that region was the Southern blot. Mod-
ifications of the Southern blot are applied to the analy-
Restriction Enzyme Cutting and Resolution
sis of RNA, proteins, and lipids in order to study gene
expression, regulation, and protein modifications (see The first step in the Southern blot procedure is diges-
Table 5.1). tion of test DNA with restriction enzymes. The choice
of enzymes used will depend on the genetic locus and
the application. For routine laboratory tests, restriction
Southern Blots
maps of the target DNA regions will have previously
The Southern blot is named for Edwin Southern, been determined, and the appropriate enzymes will have
who first reported the procedure.8 In the Southern been recommended. For other methods, such as typing
blot, genomic DNA is isolated and cut with restriction of unknown organisms or cloning, several enzymes may
enzymes. The fragments are separated by gel elec- be tested to find those that will be most informative.
trophoresis, denatured, and then transferred to a solid Ten to 50 μg of high-quality (intact) genomic DNA is
support such as nitrocellulose. In the final steps of the used for each restriction enzyme digestion for Southern
procedure, the DNA fragments are exposed to a labeled analysis. More or less DNA may be required depending
probe (complementary DNA or RNA) that is comple- on the sensitivity of the detection system, the volume and
mentary to the region of interest. The signal of the probe configurations of wells, and the abundance of the target
is detected to indicate the presence or absence (lack of DNA. In the clinical laboratory, specimen availability
signal) of the sequence in question. The original method may limit the amount of DNA available for testing. The
entailed hybridization of a radioactively labeled probe to DNA is mixed with the appropriate restriction enzyme
detect the DNA region to be analyzed. As long as there and buffer. Restriction enzymes are supplied with
is a probe of known identity, this procedure can analyze 10× buffers that are diluted 1/10 into the final reaction
any gene or gene region in prokaryotes or eukaryotes at volume. If more than one enzyme is to be used, each
the molecular level. Newer methods have replaced PCR sample must be digested separately for each enzyme
for many applications; however, Southern blots are still and buffer.
applied to the characterization of large regions (10 kb to Digestion is carried out for an extended time (3 hours
more than 100 kb). The following sections describe the or more) to allow complete cutting of all sites in the
parts of the Southern blot procedure in detail and discuss DNA sample. High-specific-activity enzymes are also
used to ensure complete cutting of every site. Incomplete with ethidium bromide and illuminated by ultraviolet
cutting will result in anomalous patterns, complicating (UV) light. Genomic DNA cut with restriction enzymes
interpretation of the Southern blot data. The fragments should produce a smear representing billions of frag-
resulting from the restriction digestions are resolved by ments of all sizes released by enzyme digestion. The
gel electrophoresis. The percentage and nature of the brightness of the DNA smears should be similar from
gel will depend on the size of the DNA region to be lane to lane, ensuring that equal amounts of DNA were
analyzed (see Chapter 4, Tables 4.1 and 4.2). As with added to all lanes. A large aggregate of DNA near the
all electrophoresis, a molecular-weight standard should top of the lane indicates that the restriction enzyme
be run with the test samples. Large fragments require activity was incomplete, preventing size analysis of the
longer runs at low voltage to get the best resolution. For sample. A smear located primarily in the lower region
example, 10,000- to 20,000-bp fragments are resolved in of the lane is a sign that the DNA is degraded. Uncut
0.7% agarose at 20 amperes for 16 hours. or degraded DNA will prevent accurate analysis. Repeat
After electrophoresis, it is important to observe the of the restriction digest will be required if uncut DNA
cut DNA. Figure 5.5 shows a 0.7% agarose gel stained is present. If an impurity in the DNA results in resis-
tance to restriction digestion or degradation of the DNA,
re-isolation or further purification of the DNA will be
required.
BgIII XbaI BamH1 HindIII
M C ⫹ C ⫹ C ⫹ C ⫹ Preparation of Resolved DNA
for Blotting (Transfer)
The goal of the Southern blot procedure is to analyze a
specific region of the sample DNA. The restriction frag-
ments containing the target sequence to be analyzed are
obviously not distinguishable in the collection of other
fragments that do not have the target sequence. Target
fragments can be detected by hybridization with a com-
plementary sequence of single-stranded DNA or RNA
labeled with a detectable signal. To achieve optimal
hydrogen bonding between the probe and its comple-
mentary sequence in the resolved sample DNA, the
double-stranded DNA fragments in the gel must be sep-
arated into single strands (denatured) and transferred to
a nitrocellulose membrane.
Depurination
Before moving the DNA fragments from the gel to the
membrane for blotting, the double-stranded DNA frag-
ments are denatured as the DNA remains in place in the
gel. Although short fragments can be denatured directly
as described in the following section, larger fragments
(greater than 500 bp) are more efficiently denatured
if they are depurinated before denaturation (Fig. 5.6).
FIGURE 5.5 Properly restricted genomic DNA will produce
a smear of fragments along the agarose gel lane ranging in size Therefore, for large fragments, the gel is first soaked in
depending on the frequency of the particular enzyme restric- dilute hydrogen chloride (HCl) solution, a process that
tion sites in the DNA. Among these are the fragments coming removes purine bases from the sugar-phosphate back-
from the region under study. After probing, only those frag- bone. This will “loosen up” the larger fragments for
ments will be visible. more complete denaturation.
The binding interaction is hydrophobic and electrostatic

between the negatively charged DNA and the positive
T A charges on the membrane. Nitrocellulose-based mem-
branes bind 70 to 150 μg of nucleic acid per square
centimeter. Membrane pore sizes (0.05 to 0.45 μm) are
suitable for DNA fragments from a few hundred bases
C G up to those greater than 20,000 bp in length.
Advanced Concepts
G C
Treatment of DNA with dilute (0.1 to 0.25 mM)
hydrochloric acid results in hydrolysis of the gly-
cosidic bonds between purine bases and the sugar
A T of the nucleotides. This loss of purines (adenines
and guanines) from the sugar-phosphate backbone
of DNA leaves apurinic sites (see Fig. 5.6). The
DNA backbone remains intact and holds the rest
of the bases in linear order. Removal of some of
FIGURE 5.6 An apurinic site in double-stranded DNA. Loss the purine bases facilitates the subsequent break-
of the guanine leaves an open site but does not break the sug- ing of hydrogen bonds between the two strands of
ar-phosphate backbone of the DNA. the DNA during the denaturation step in Southern
blotting.
Denaturation
DNA is denatured by exposing the gel to a strong base Pure nitrocellulose has a high binding capacity for pro-
such as sodium hydroxide (NaOH). NaOH promotes teins as well as nucleic acids. It is the most versatile
breakage of the hydrogen bonds holding the DNA medium for molecular transfer applications. It is also
strands to one another. The resulting single strands are compatible with different transfer buffers and detection
then available to hydrogen bond with the single-stranded systems. Nitrocellulose is not as sturdy as other media
probe. Further, the single-stranded DNA will bind more and becomes brittle upon drying. Because all genomic
tightly than double-stranded DNA to the nitrocellulose fragments are permanently bound to the membrane, the
membrane upon transfer. hybridized probe can be stripped, and a second probe
can be hybridized to the same membrane. Reinforced
Blotting (Transfer) nitrocellulose is more appropriate for applications where
multiple probings may be necessary. Mechanically stable
Before exposing the denatured sample DNA to the
membranes can be formulated with a net neutral charge
probe, the DNA is transferred, or blotted, to a solid
to decrease nonspecific binding. These membranes have
substrate that will facilitate probe binding and signal
a very high binding capacity (less than 400 μg/cm2),
detection. This substrate is usually nitrocellulose, nylon,
which increases the test sensitivity. A covalent attach-
or cellulose modified with a diethyl aminoethyl, or a
ment of nucleic acid to these membranes is achieved
carboxymethyl (CM) chemical group. Membranes of
by exposure of the DNA on the membrane to UV-light
another type, polyvinyl difluoride (PVDF), are used
cross-linking.
for immobilizing proteins for probing with antibodies
Membranes with a positive charge more effectively
(western blots).
bind small fragments of DNA. These membranes,
Membrane Types however, are more likely to retain protein or other con-
Single-stranded DNA avidly binds to nitrocellulose mem- taminants that will contribute to background noise after
branes with a noncovalent, but irreversible, connection. the membrane is probed.
Before transfer of the sample, membranes are moist- towels are stacked on top of the membrane. The buffer
ened by floating them on the surface of the transfer will move by capillary action from the lower reservoir to
buffer. Any dry spots (areas where the membrane does the dry material on top of the gel. The movement of the
not properly hydrate) will remain white while the rest buffer transversely through the gel will carry the dena-
of the membrane darkens with buffer. If the membrane tured DNA out of the gel. When the DNA contacts the
does not hydrate evenly, dry spots will inhibit binding nitrocellulose membrane, the DNA will bind to it.
of the sample.
Advanced Concepts
Advanced Concepts
Diethylaminoethyl (DEAE)-conjugated cellu-
Binding of single-stranded DNA to nitrocellulose lose effectively binds nucleic acids and neg-
does not prevent hydrogen-bond formation of the atively charged proteins. PVDF and charged
immobilized DNA with complementary sequences. carboxymethyl cellulose membranes are used only
The bond between the membrane and the DNA is for protein (western) blotting. These membranes
much stronger than the hydrogen bonds that hold bind nucleic acid and proteins by hydrophobic
complementary strands together. This allows for and ionic interactions with a binding capacity of
removal of probes and re-probing of different 20 to 40 μg/cm2 to 150 μg/cm2 for PVDF. Modi-
target regions. fications of PVDF are designed to optimize use in
a variety of test and sample types. These include
small-pore-size membranes for use with small
proteins and membranes optimized for fluorescent
Transfer Methods
detection.
Transfer of nucleic acid to protein is performed in several
ways. The goal is to move the DNA from the gel to a
membrane substrate for probing. The membrane must Capillary transfer is simple and relatively inex-
be equilibrated in the transfer buffer before coming into pensive. No instruments are required. The transfer,
contact with the DNA in the gel. Membranes should be however, can be less than optimal, especially with large
handled carefully, preferably with powder-free gloves, gels. Bubbles, salt crystals, or other particles between
avoiding folding or creasing of the membrane. The orig- the membrane and the gel can cause loss of information
inal method developed by Southern used capillary trans- or staining artifacts. The procedure is also slow, taking
fer (Fig. 5.7). For capillary transfer, the gel is placed from a few hours to overnight for large fragments.
on top of a reservoir of buffer, which can be a shallow A second method, called electrophoretic transfer,
container or filter papers soaked in high-salt buffer, for uses electric current to move the DNA from the gel
example, 10× saline sodium citrate (10X SSC: 1.5 M to the membrane (Fig. 5.8). This system utilizes elec-
NaCl, 0.15 M Na citrate) or commercially available trodes attached to membranes above (anode) and below
transfer buffers. The nitrocellulose membrane is placed (cathode) the gel. The current carries the DNA trans-
directly on the gel, and dry absorbent filter paper or paper versely from the gel to the membrane. Electrophoretic
Dry paper
Nitrocellulose
membrane
Gel FIGURE 5.7 Capillary transfer. Driven by capillary

movement of buffer from the soaked paper to the dry
Soaked paper paper, denatured DNA moves from the gel to the mem-
brane. The DNA will adhere to the membrane, which
Buffer
will be subsequently exposed to the probe.
Whatman Nitrocellulose
paper Gel membrane
– +
Buffer Support Glass Buffer

plates
FIGURE 5.8 Electrophoretic transfer. This system uses electric current to move the DNA transversely through the gel to the
membrane. This type of transfer is used mostly for small fragments or proteins.
Gel
FIGURE 5.9 Vacuum transfer. This system uses Nitrocellulose

suction and buffer recirculation to move the DNA out membrane
of the gel and onto the membrane. Vacuum transfer is Porous plate
generally faster than capillary transfer for large DNA
fragments; however, unlike capillary transfer, vacuum Recirculating buffer
transfer requires specialized equipment. Vacuum
transfer is performed in a “tank” or by a “semidry” oven (80°C, 30 to 60 minutes) or by UV cross-linking,

approach. In the tank method, the electrodes transfer that is, covalently attaching the DNA to the nitrocel-
current through the gel and membrane through electro- lulose using UV-light energy. Baking or cross-linking
phoresis buffer, as shown in Figure 5.8. In the semidry covalently attaches the DNA to the membrane and pre-
method, the electrodes contact the gel-membrane sand- vents the DNA fragments from washing away or moving
wich directly, requiring only enough buffer to soak the on the membrane during extended procedures or when
gel and membrane. The tank electrophoretic transfer is sequential probings are to be done.
preferred for large proteins resolved on acrylamide gels,
whereas the semidry method is frequently used for small
proteins. PROBE HYBRIDIZATION
Vacuum transfer is a third method of DNA blotting
(Fig. 5.9). This blotting technique uses suction to move Following immobilization of the DNA, a prehybridiza-
the DNA from the gel to the membrane in a recirculating tion step is required to prevent the probe from binding to
buffer. Like electrophoretic transfer, this method trans- nonspecific sites on the membrane surface, which may
fers the DNA more rapidly than capillary transfer—in cause background noise. Prehybridization involves
2 to 3 hours rather than overnight. Also, it avoids dis- incubating the membrane in the same buffer in which the
continuous transfer due to air trapped between the mem- probe will subsequently be introduced or in a specially
brane and the gel. One disadvantage of the second and formulated prehybridization buffer solution. At this
third methods is the expense and maintenance of the point, the buffer does not contain probe. Prehybridization
electrophoresis and vacuum equipment. buffer consists of such blocking agents as Denhardt
After transfer, the cut, denatured DNA is avidly solution (Ficoll, polyvinyl pyrrolidine, bovine serum
bound to the membrane. The DNA can be permanently albumin) and salmon sperm DNA. Sodium dodecyl
immobilized to the membrane by baking in a vacuum sulfate (SDS, 0.01%) may also be included, along with
formamide, the latter especially for RNA probes. The of the RNA to nitrocellulose. This is accomplished by
membrane is exposed to the prehybridization buffer at rinsing the gel in deionized water. RNA is transferred
the optimal hybridization temperature for 30 minutes in 10× or 20× SSC or 10× SSPE (1.8 M NaCl, 0.1 M
to several hours, depending on the specifications in the sodium phosphate, pH 7.7, 10 mM EDTA) to nitrocel-
protocol. At this stage, the sample is ready for hybridiza- lulose as described previously for DNA. For small tran-
tion with the probe, which will allow visualization of the scripts (500 bases or less), 20× SSC should be used.
specific gene or region of interest. The blotting procedure for RNA in the northern blot
is performed in 20× SSC, similar to the procedure for
DNA transfer in the Southern blot. Prehybridization and
Northern Blots
hybridization in formamide/SSC/SDS prehybridization/
The northern blot technique, a modification of the hybridization buffers are also performed as with South-
Southern blot technique, was designed to investigate ern blot. If the RNA has been denatured in glyoxal, the
RNA structure and quantity. Although most northern membrane must be soaked in warm Tris buffer (65°C)
analyses were performed to investigate levels of gene to remove any residual denaturant immediately before
expression (transcription from DNA) and stability, the prehybridization.
analyses were also used to investigate RNA structural Although it is still used in some research applica-
abnormalities resulting from aberrations in synthesis or tions, the northern blot has been mostly replaced by
processing, such as alternative splicing. Splicing abnor- more efficient technologies with lower sample demands.
malities are responsible for a number of diseases, such as Genomic technologies, such as arrays (described later in
beta-thalassemias and familial isolated growth hormone this chapter), provide more comprehensive information
deficiency. Analysis of RNA structure and quantity indi- with less demand on RNA length.
rectly reveals mutations in the regulatory or splicing
signals in DNA.
Western Blots
To perform a northern blot, nucleic acid isolation
methods for RNA are used. An RNase-free environment Another modification of the Southern blot is the western
must always be maintained for RNA preparation. After blot.9 The immobilized target for a western blot is
isolation and quantification of RNA, the samples (up to protein. There are many variations on western blots.
approximately 30 μg total RNA or 0.5 to 3.0 μg polyA Generally, serum, cell lysate, or extract is separated
RNA, depending on the relative abundance of the tran- on SDS-polyacrylamide gels (SDS-PAGE) or isoelec-
script under study) are applied directly to agarose gels. tric focusing gels (IEF). The former resolves proteins
Agarose concentrations of 0.8% to 1.5% are usually according to molecular weight, and the latter resolves
employed. Polyacrylamide gels may also be used, espe- proteins according to charge. Dithiothreitol or 2-mer-
cially for smaller transcripts—for instance, for analysis captoethanol may also be used to separate proteins into
of viral gene expression. Gel electrophoresis of RNA subunits. Polyacrylamide concentrations vary from 5%
is carried out under denaturing conditions for accurate to 20%. Depending on the complexity of the protein and
transcript size assessment. Complete denaturation is also the quantity of the target protein, 1 to 50 μg of protein
required for efficient transfer of the RNA from the gel to is loaded per well. Before loading, the sample is treated
the membrane, as with the transfer of DNA in the South- with denaturant, such as mixing 1:1 with 0.04 M Tris
ern blot. Because the denaturation is maintained during HCl, pH 6.8, 0.1% SDS. The accuracy and sensitivity
electrophoresis, a separate denaturation step is not of the separation can be enhanced by using a combina-
required for northern blots. After electrophoresis, repre- tion of IEF gels followed by SDS-PAGE or by using
sentative lanes can be cut from the gel, soaked in ammo- two-dimensional gel electrophoresis. Prestained molec-
nium acetate to remove the denaturant, and stained with ular-weight standards are run with the samples to orient
acridine orange or ethidium bromide to assess quality the membrane after transfer and to approximate the sizes
and equivalent sample loading. of the proteins after probing. Standards ranging from
Denaturant such as formaldehyde must be removed 11,700 d (cytochrome C) to 205,000 d (myosin) are
from the gel before transfer because it inhibits binding commercially available.
The gel system used may affect subsequent probing

of proteins with antibodies. Specifically, denaturing Advanced Concepts
gels could affect epitopes (antigenic sites on the protein)
such that they will not bind with the labeled antibodies. A frequent application of the western blot method
Gel pretreatment with mild buffers, such as 20% glyc- is confirmation of enzyme-linked immunoassay
erol in 50 mM Tris-HCl, pH 7.4, can renature proteins (ELISA) results for human immunodeficiency
before transfer. virus (HIV) and hepatitis C virus (HCV), among
After electrophoresis, proteins are blotted to mem- other microbes. In this procedure, known viral
branes by capillary or electrophoretic transfer. Nitro- proteins are separated by electrophoresis and
cellulose has a high affinity for proteins and is easily transferred and bound to a nitrocellulose mem-
treated with detergent (0.1% Tween 20 in 0.05 M Tris brane. The patient’s serum is overlaid on the
and 0.15 M sodium chloride, pH 7.6) with 5% dry milk membrane, and antibodies with specificity to viral
to prevent binding of the primary antibody probe proteins bind to their corresponding protein anti-
to the membrane itself (blocking) before hybridiza- gens. Unbound patient antibodies are washed off,
tion. Binding of proteins to nitrocellulose is probably and binding of antibodies is detected by adding a
hydrophobic because nonionic detergents can remove labeled antihuman immunoglobulin antibody. If
proteins from the membrane. Other membrane types viral antibodies are present in the patient’s serum,
used for protein blotting are PVDF and anion (DEAE) they can be detected with antihuman antibody
or cation (CM) exchange cellulose. After incubation probes appearing as a dark band on the blot corre-
with the primary antibody for 12 to 16 hours, the blot sponding to the specific HIV protein to which the
is washed in the same buffer (without dry milk) and antibody is specific.
incubated with the secondary antibody conjugated with
enzyme. The blot is washed again to remove excess
secondary antibody conjugate, and the chemilumines- Probes for western blots are specific binding proteins
cent or color signal is developed with the addition of or antibodies. A labeled secondary antibody directed
substrate. against the primary binding protein is then used for the
visualization of the protein band of interest.
PROBES DNA Probes

The probe for Southern and northern blots is a single- DNA probes are created in several ways. In early
stranded fragment of nucleic acid attached to a signal- methods, a fragment of the gene to be analyzed was
producing moiety. The purpose of the probe is to cloned on a bacterial plasmid and then isolated by
identify one or more sequences of interest within a large restriction enzyme digestion and gel purification. The
amount of nucleic acid. The probe therefore should fragment, after labeling (see following discussion) and
hybridize specifically with the target DNA or RNA that denaturation, was then used in Southern or northern blot
is to be analyzed. The probe can be RNA, denatured procedures.
DNA, or other modified nucleic acids. Peptide nucleic Other sources of DNA probes include the isolation of
acids (PNAs) and locked nucleic acids have also been a sequence of interest from viral genomes and in vitro
used as probes. These structures contain normal nitrogen organic synthesis of nucleic acid of a predetermined
bases that can hybridize with complementary DNA or sequence. The latter is used only for short, oligomeric
RNA, but the bases are connected by backbones differ- probes. Probes may also be synthesized using the poly-
ent from the natural phosphodiester backbone of DNA merase chain reaction (PCR).
and RNA. These modified nucleic acids are resistant to The length of the probe will, in part, determine the
nuclease degradation and, because of a reduced negative specificity of the hybridization reaction. Probe lengths
charge on their backbone, can hybridize more readily to range from tens to thousands of base pairs. In an anal-
target DNA or RNA. ysis of the entire genome in a Southern blot, longer
probes are more specific for a DNA region because they complementary to the target sequence. A probe of iden-
must distinguish among many closely similar sequences. tical sequence to the target RNA (coding sequence) will
Shorter probes are not usually used in Southern blots not hybridize.
because short sequences are more likely to be found in RNA probes are labeled to produce a signal by incor-
multiple locations in the genome, resulting in high back- porating a radioactive or modified nucleotide during the
ground binding to sequences not related to the target in vitro transcription process. Labeling during synthe-
region of interest. sis provides RNA probes with a high specific activity
The probe is constructed so that it has a complemen- (signal to micrograms of probe) that increases the sen-
tary sequence to the targeted gene. In order to bind to sitivity of the probe. To avoid background noise, some
the probe, then, the target nucleic acid has to contain protocols include digestion of nonhybridized probe,
the sequence of interest. Properly prepared and stored using a specific RNase such as RNase A, after hybrid-
DNA probes are relatively stable. Double-stranded DNA ization is complete.
probes must be denatured before use. This is usually RNA probes are generally less stable than DNA
accomplished by heating the probe (e.g., 95°C, 10 to probes and cannot be stored for long periods. Synthesis
15 min) in hybridization solution or treating with 50% of an RNA probe by transcription from a stored tem-
formamide/2× SSC at a lower temperature for a shorter plate is relatively simple and is easily performed within
time (e.g., 75°C, 5 to 6 min). a few days of use. The DNA template can be removed
from the probe by treatment with RNase-free DNase.
Although RNA is already single stranded, denaturation
RNA Probes
before use is recommended in order to eliminate the sec-
RNA probes are often made by transcription from a syn- ondary structure internal to the RNA molecule.
thetic DNA template in vitro. These probes are similar
to DNA probes with equal or greater binding affinity
Other Nucleic Acid Probe Types
to complementary sequences. Because RNA and DNA
form a stronger helix than DNA/DNA, the RNA probes Peptide nucleic acid, locked nucleic acid, and unlocked
may offer more sensitivity than DNA probes in the nucleic acid probes (Figs. 5.10 and 5.11) are synthesized
Southern blot. using chemical methods.10 These modified nucleic acids
RNA probes can be synthesized directly from a have the advantage of being resistant to nucleases that
plasmid template or from template DNA produced by degrade DNA and RNA by breaking the phosphodiester
PCR. Predesigned systems commercially available for backbone. Further, the negative charge of the phospho-
this purpose include plasmid vector DNA containing a diester backbones of DNA and RNA counteract hydro-
binding site for RNA polymerase (promoter), a cloning gen bonding between the bases of the probe and target
site for the sequences of interest, and a DNA-dependent sequences. Structures such as peptide nucleic acid (PNA)
RNA polymerase from Salmonella bacteriophage SP6 or that do not have a negative charge hybridize more effi-
Escherichia coli bacteriophage T3 or T7. DNA sequences ciently. Unlocked nucleic acids (UNA) lack the C2′–C3′
complementary to the RNA transcript to be analyzed are ribose sugar bond found in ribonucleoside, resulting in
cloned into the plasmid vector using restriction enzymes. high flexibility. Incorporation of UNA nucleotides desta-
The recombinant vector containing the gene of interest bilizes the double helix. UNA can therefore “fine-tune”
is then linearized, and the RNA probe is transcribed in the hybridization of probes to targets, especially in short
vitro from the promoter. The coding strand transcript sequences.11
can be used for Southern blots. For northern blots, the
antisense transcript (complementary to the coding strand
Protein Probes
transcript on the blot) is generated by inverted placement
of the fragment so that the promoter starts at the end Western blot protein probes are antibodies that bind spe-
of the gene. Either coding or complementary RNA will cifically to the immobilized target protein. Polyclonal or
hybridize to a double-stranded DNA target. RNA probes monoclonal antibodies are used for this purpose. Poly-
for northern blots must be designed so that the probe is clonal antibodies are products of a generalized response
OR OR
–
NH2
O P O O– P O
Base
O O
O Base O Base
N
O
FIGURE 5.10 Peptide nucleic acids have O

NH O
the phosphodiester bond (left) replaced with O O
carbon-nitrogen peptide bonds (center). R
O– P O O– P O
Locked nucleic acids are bicyclic nucleoside
monomers where the ribose sugar contains a O O
methylene link between its 2′ oxygen and 4′
carbon atoms (right). R R
NH2 NH2
N N
N
HO O O HO ON N
O N3'-phosphoramidate NH2
O H3C O NH
–O
NH N
Phosphodiester P
P N
O N O –O O O O
O O
NH2 O
O N –O O OR N
H3C N NH
Methylphosphonate P 2'-O-alkyl RNA P
O O ON N O O ON N NH2
O
O N H3C
N N NH
–S O NH P
Phosphorothioate P O O N O
O
O O ON N NH2 Morpholino
phosphorodiamidate NH2
NH2 N
H3B O N N
N
P O N N
Borane phosphonate N
O O ON N
H N
Peptide nucleic acid O
–O O
P O NH2
3'-O-phosphopropylamino
O O NH3
OH
FIGURE 5.11 Modifications of the phosphodiester backbone of nucleic acids include substitution on the alpha-phosphate group
with alkyl, sulfur, or other groups. The ribose may also have modifications with nitrogen or alkyl groups or replacement with
peptide rather than phosphodiester bonds (right).
to a specific antigen, usually a peptide or protein. Small the antibody titer by slowing the degradation of the
molecules (haptens) attached to protein carriers, car- protein and lengthening the time the immune system is
bohydrates, nucleic acids, and even to whole cells and exposed to the stimulating antigen. Specific immuno-
tissue extracts can be used to generate an antibody globulins are subsequently isolated from sera by affinity
response. Adjuvants, such as Freund’s adjuvant, enhance chromatography.
Polyclonal antibodies are comprised of a mixture Probe Labeling

of immunoglobulins directed at more than one epitope
(molecular structure) on the antigen. Monoclonal anti- In order to visualize the probe bound to target fragments
bodies are more difficult to produce. Kohler and Mil- on a membrane, the probe must be labeled and generate
stein first demonstrated that spleen cells from immunized a detectable signal. In the past, classical Southern analy-
mice could be fused with mouse myeloma cells to form sis methods called for radioactive labeling with 32P. This
hybrid cells (hybridomas) that could grow in culture labeling was achieved by introduction of nucleotides
and secrete antibodies.12 By cloning the hybridomas containing radioactive phosphorus to the probe. Today,
(growing small cultures from single cells), preparations many medical laboratories now use nonradioactive label-
of specific antibodies could be produced continuously. ing to avoid the hazard and expense of working with
The clones could then be screened for antibodies that radiation. Nonradioactive labeling methods are based on
best react with the target antigen. The monoclonal anti- indirect detection of a tagged nucleotide incorporated in
bodies are isolated from cell culture fluid; higher titers of or added to the probe. The two most commonly used
antibodies are obtained by inoculating the antibody-pro- nonradioactive tags are biotin and digoxigenin (Fig.
ducing hybridoma into mice and collecting the perito- 5.12), either of which can be attached covalently to a
neal fluid. The monoclonal antibody is then isolated by nucleotide triphosphate, usually UTP or CTP.
chromatography. There are three basic methods that are used to label a
Polyclonal antibodies are useful for immunoprecipi- DNA probe: end labeling, nick translation, and random
tation methods and for western blots. With their greater priming. In end labeling, labeled nucleotides are added
specificity, monoclonal antibodies can be used for almost to the end of the probe using terminal transferase or T4
any procedure. In western blot technology, polyclonal polynucleotide kinase. In nick translation, the labeled
antibodies can give a more robust signal, especially if nucleotides are incorporated into single-stranded breaks,
the target epitopes are partially lost during electrophore- or nicks, that are substrates for nucleotide addition by
sis and transfer. Monoclonal antibodies are more specific DNA polymerase. The polymerase uses the intact com-
and may give less background noise; however, if the tar- plementary strand for a template and displaces the previ-
geted epitope is lost, these antibodies do not bind, and ously hybridized strand as it extends the nick. Random
no signal is generated. The dilution of primary antibody priming generates new single-stranded versions of the
can range from 1/100 to 1/100,000, depending on the probe with the incorporation of the labeled nucleotides.
sensitivity of the detection system. The synthesis of these new strands is primed by oligo-
mers of random sequences that are 6 to 10 bases in
Advanced Concepts length. These short sequences will, at some frequency,
complement sequences in the denatured probe and prime
Alternative nucleic acids are not only useful in the synthesis of copies of the probe sequences with incorpo-
laboratory, but they are also potentially valuable in rated labeled nucleotides.
the clinic. Several structures have been proposed RNA probes are transcribed from cloned DNA or
for use in antisense gene therapy (Fig. 5.11). Intro- amplified DNA. These probes are labeled during their
duction of sequences complementary to messenger synthesis with radioactive, biotinylated, or digoxigen-
RNA of a gene (antisense sequences) will prevent in-tagged nucleotides. Unlike double-stranded com-
translation of that mRNA and expression of that plementary DNA probes and targets that contain both
gene. If this could be achieved in whole organ- strands of the complementary sequences, RNA probes
isms, selected aberrantly expressed genes or even are single stranded, with only one strand of the comple-
viral genes could be turned off. One drawback of mentary sequence represented.
this technology is the degradation of natural RNA
and DNA by intracellular nucleases. The nucle-
Nucleic Acid Probe Design
ase-resistant structures are more stable and avail-
able to hybridize to the target mRNA. The most critical parts of any hybridization procedure
are the design and optimal hybridization of the probe,
O
HN NH
O
HO
CH3
X
S CH3
OH
O O
C
O O O CH2
CH CH CH2 NH C (CH2)5 NH C CH2
HN
O N
O
LiO P O H 2C O
OLi 3
OH
FIGURE 5.12 Biotin (top) has a variable side chain (X). The polycyclic digoxigenin (bottom) is shown covalently attached to
UTP (dig-11-UTP). This molecule can be covalently attached or incorporated into DNA or RNA to make a labeled probe.
both of which determine the specificity of the results.

With nucleic acids, the more optimal the hybridization Label
conditions for a probe/target interaction, the more spe-
Secondary antibody
cific the probe. Longer probes (500 to 5,000 bp) offer
greater specificity with decreased background noise Target protein Primary antibody
because they are less affected by point mutations or
polymorphisms within the target sequence or within the
probe itself. Long probes, however, may be difficult or
expensive to synthesize.
Shorter probes (less than 500 bp) are less specific
than longer ones in Southern blotting applications. A
FIGURE 5.13 Probe binding to western blots may
short sequence has a higher chance of being repeated include an unlabeled primary antibody that is bound by
randomly in unrelated regions of the genome. Short a secondary antibody carrying a label for detection.
probes are ideal, however, for mutation analysis because
their binding affinity is sensitive to single-base-pair
usually horseradish peroxidase (HRP) or alkaline
changes within a target binding sequence.
phosphatase. When exposed to a light- or col-
or-generating substrate, the enzyme will produce a
detectable signal on the membrane or on an auto-
Advanced Concepts radiogram. Unconjugated antibodies are detected
after binding with a conjugated secondary antibody
The protein probes used in western blot appli- to the primary probe, such as mouse antihuman or
cations may be labeled with 35S for radioactive rabbit antimouse antibodies (Fig. 5.13). The sec-
detection. For nonradioactive detection, western ondary antibodies will recognize any primary anti-
protein probes are covalently bound to an enzyme, body by targeting the FC region.
The probe sequence can affect its binding performance. DS

A probe with internal complementary sequences will
fold and hybridize with itself, which will compete with
hybridization to the intended target. The probe folding DS=SS
or secondary structure is especially strong in sequences
with high GC content, decreasing the binding effi-
ciency to the target sequence and, therefore, the test SS
sensitivity. Tm
Increasing temperature
HYBRIDIZATION CONDITIONS, STRINGENCY FIGURE 5.14 Melting temperature, Tm, is the point at which
exactly half of a double-stranded sequence becomes single
stranded. The melting temperature is determined at the inflec-
Southern blot and northern blot probing conditions must
tion point of the melt curve. DS, double stranded; SS, single
be empirically optimized for each nucleic acid target. stranded.
Stringency is the combination of conditions under which
the target is exposed to the probe. Conditions of high
stringency are more demanding of probe/target com- hybrids, therefore, are slightly different from the formula
plementarity and length. Low-stringency conditions are for a DNA:DNA helix. RNA:DNA hybrids increase the
more forgiving. If conditions of stringency are set too Tm by 10°C to 15°C. RNA:RNA hybrids increase the Tm
high, the probe will not bind to its target. If conditions by 20°C to 25°C. The melting temperature will also be
are set too low, the probe will bind unrelated targets, different if PNA or other alternative nucleic acids are
complicating the interpretation of the final results. used as probes.13
Several factors affect stringency. These include the The Tm is also a function of the extent of complemen-
temperature of hybridization; the salt concentration of tarity between the sequence of the probe and that of the
the hybridization buffer; and the concentration of dena- target sequence. For each 1% difference in sequence, the
turant, such as formamide, in the buffer. The length and Tm decreases 1.5°C.
nature of the probe sequence can also influence the level As the length of probes decreases, the sequence
of stringency. A long probe or one with a higher per- becomes more influential for the final Tm. The Tm for
centage of G and C bases will bind under more strin- short probes (14 to 20 bases) can be estimated by a
gent conditions than a short probe or one with greater simpler formula:
numbers of A and T bases will bind. The ideal hybridiza- Tm = 4°C × number of GC pairs +
tion conditions are estimated from the calculation of the 2°C × number of AT pairs
melting temperature, or Tm, of the probe sequence. The
Tm is a way to express the amount of energy required to The hybridization temperature of oligonucleotide probes
separate the hybridized strands of a given sequence (Fig. is about 5°C below the melting temperature.
5.14). At the Tm, half of the sequence is double stranded, The effect of sequence complexity on hybridization
and half is single stranded. One formula for the Tm of a efficiency can be illustrated by the Cot value. Sequence
double-stranded DNA sequence in solution is complexity is the length of unique (nonrepetitive) nucle-
otide sequences in a genome, chromosome, or other
Tm = 81.5°C + 16.6 log M + 0.41(%G + C) set of double-stranded sequences. After denaturation,
− 0.61(% formamide) − (600 / n ) complex sequences require more time to reassociate
where M = sodium concentration in mol/L, and n = than simple sequences, such as polyA:polyU. Cot is an
number of base pairs in the shortest duplex. expression of the sequence complexity (Fig. 5.15). Cot
RNA:RNA hybrids are more stable than DNA:DNA is equal to the initial DNA concentration (Co) times the
hybrids due to less constraint by the RNA phosphodi- time required to reanneal it (t). Cot1/2 is the time required
ester backbone. DNA:RNA hybrids have intermediate for half of a double-stranded sequence to anneal under a
affinity. The formulae for RNA:RNA or DNA:RNA given set of conditions.
1bp
Size
10,000 bp
Hybridizations are generally performed in hybridiza-
Complexity tion bags or in glass cylinders. Within limits, the sen-
DS sitivity of the analysis increases with increased probe
concentration. Because the probe is the limiting reagent,
it is practical to keep the volume of the hybridization
DS=SS
solution low. The recommended volume of hybridization
buffer is approximately 10 mL/100 cm2 of membrane
surface area.
Formamide in the hybridization buffer effectively
SS
–6 –5 –4 –3 –2 –1 0 1 lowers the optimal hybridization temperature. This
Log Cot is especially useful for RNA probes and targets that,
because of secondary structure, are more difficult to
FIGURE 5.15 Reannealing of single-stranded (SS) DNA to denature and tend to have a higher renaturation (hybrid-
double-stranded (DS) DNA versus time at a constant concen- ization) temperature. Incubation of the hybridization
tration yields a sigmoid curve. The complexity of the DNA system in sealed bags in a water bath or in capped glass
sequence will widen the sigmoid curve. Increasing the length
cylinders in rotary ovens maintains the entire membrane
of the double-stranded DNA will shift the curve to the right.
surface area at a constant temperature.
Short probes (less than 20 bases) can hybridize in
1 to 2 hours. In contrast, longer probes require much
Advanced Concepts longer hybridization times. For Southern and northern
blots with probes greater than 1,000 bases in length,
Cot was used to demonstrate that mammalian DNA incubation is carried out for 16 hours or more. Raising
consisted of sequences of varying complexity. the probe concentration can increase the hybridization
Britten and Kohne14 used E. coli and calf thymus rates. Also, inert polymers, such as dextran sulfate,
DNA to demonstrate this complexity. When they polyethylene glycol, or polyacrylic acid, accelerate the
measured reassociation of E. coli DNA versus hybridization rates for probes longer than 250 bases.
time, a sigmoid curve was observed, as expected
for DNA molecules with equal complexity. In com-
parison, the calf DNA reassociation was multifac- Advanced Concepts
eted and spanned several orders of magnitude (see
Fig. 5.15). The spread of the curve resulted from The nature of the probe label will affect hybrid-
the mixture of slowly renaturing unique sequences ization conditions. Unlike 32P labeling, the bulky
and rapidly renaturing repeated sequences (satel- nonradioactive labels (see Fig. 5.12) disturb the
lite DNA), both of which are components of mam- hybridization of the DNA chain. The temperature
malian DNA. of hybridization with these types of probes will
be lower than that used for radioactively labeled
probes.
Tm and Cot values can provide a starting point for opti-
mizing the stringency conditions for Southern or north-
ern blot analysis. Hybridization at a temperature 25°C DETECTION SYSTEMS
below the Tm for 1 to 3 Cot1/2 is considered optimal for
a double-stranded DNA probe. The final conditions must After the transfer of gel-separated DNA, RNA, or
be established empirically, especially for short probes. protein to a solid membrane support and hybridization
The stringency conditions for routine analyses, once or binding of a specific probe to the target sequence
established, are used for all subsequent assays. In the of interest, the next step is to detect whether the probe
event that a component of the procedure is altered, new has bound to the immobilized target. 32P-labeled probes
conditions may have to be established. offered the advantages of simple and sensitive detection.
Radioactive isotope probe Anti-digoxigenin or streptavidin

conjugated to alkaline phosphatase
Digoxigenin or biotin
Probe
Nitrocellulose
membrane
Nitrocellulose
membrane
Autoradiograph
X-ray film Autoradiography
X-ray film
FIGURE 5.16 A DNA or RNA probe labeled with radioactive

phosphorous atoms (32P or 33P) hybridized to target (homolo-
gous) sequences on a nitrocellulose membrane. The fragments
to which the probe is bound can be detected by exposing auto-
radiography film to the membrane. FIGURE 5.17 Indirect nonradioactive detection. The probe is
covalently attached to digoxigenin or biotin. After hybridiza-
tion, the probe is bound by antibodies to digoxigenin or
streptavidin conjugated to alkaline phosphatase (AP). This
After hybridization, unbound probe is washed off, and complex is exposed to color- or light-producing substrates of
the blot is exposed to light-sensitive film to detect the AP, producing color on the membrane or light detected with
fragments that are hybridized to the radioactive probe autoradiography film.
(Fig. 5.16). The wash conditions must be formulated so
that only completely hybridized probe remains on the away. Then, anti-digoxigenin antibody or streptavi-
blot. Typically, the wash conditions are more stringent din, respectively, conjugated to alkaline phosphatase
than those used for hybridization. (AP conjugate; Fig. 5.17) is added to the reaction mix
Nonradioactive detection systems require a more to bind to the digoxigenin- or biotin-labeled probe:tar-
involved detection procedure. For most nonradioactive get complex. HRP conjugates may also be used in this
systems, the probe is labeled with a nucleotide cova- procedure. After the binding of the conjugate and the
lently attached to either digoxigenin or biotin. The washing away of unbound conjugate, the membrane is
labeled nucleotide is incorporated into the nucleotide bathed in a solution of substrate that, when dephosphor-
chain of the probe by in vitro transcription, nick trans- ylated by AP or oxidized by HRP, produces a signal.
lation, primer extension, or addition by terminal trans- Substrates frequently used are dioxetane or tetrazolium
ferase. After a digoxigenin- or biotin-labeled probe is dye derivatives, which generate chemiluminescent
hybridized with the blot with sample(s) containing the (Fig. 5.18) or color (Fig. 5.19) signals, respectively (see
target sequence of interest, unbound probe is washed Table 5.2).
O O O O *
OCH3 PO3 OCH3 O OCH3
Alkaline O–
O O O–
phosphatase + Light
PO3=
FIGURE 5.18 Light is emitted from 1,2-dioxetane substrates after dephosphorylation by alkaline phosphatase to an unstable
structure. This structure releases an excited anion that emits light.
O
O
O P O–
Cl O P O– Cl OH Cl
O– O
Br Br Br HN
O–
N– Phosphatase NH NH Br
O Cl
BCIP
(colorless, soluble) Oxidation Blue precipitate
Reduction
OCH3 OCH3
N H2CO OCH2
N
N N N N
N
N N N N N N N
NH HN
NO2 NO2
O2N
NO2
NBT Blue precipitate
(yellowish, soluble)
FIGURE 5.19 Generation of color with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Alkaline
phosphatase dephosphorylates BCIP, which then reduces NBT, making an insoluble blue precipitate.
Advanced Concepts
Advanced Concepts
Optimization may not completely eliminate all
nonspecific binding of the probe. This will result There are several substrates for chemilumines-
in extra bands in control lanes due to cross-hy- cent detection that are 1,2-dioxetane derivatives,
bridizations. At a given level of stringency, any such as 3-(2′-spiroadamantane)-4-methoxy-4-(3′-
increase to eliminate cross-hybridization will phosphoryloxy) phenyl-1,2-dioxetane (AMPPD).
lower the binding to the intended sequences. It Dephosphorylation of these compounds by the
becomes a matter of balancing the optimal probe alkaline phosphatase conjugate bound to the probe
signal with the least amount of nontarget binding. on the membrane results in a light-emitting product
Cross-hybridizations are usually recognizable as (see Fig. 5.18). Other luminescent molecules
bands of the same size in multiple runs. Cross- include acridinium ester and acridinium (N-sulfo-
hybridization bands are taken into account in the nyl) carboxamide labels, isoluminol, and electro-
final interpretation of the assay results. chemiluminescent ruthenium trisbipyridyl labels.
TABLE 5.2 Nonradioactive Detection Systems
Type of Detection Enzyme Reagent Reaction Product
Chromogenic HRP 4-chloro-1-naphthol (4CN) Purple precipitate
HRP 3,3′-diaminobenzidine Dark brown precipitate
HRP 3,3′,5,5′,-tetramethylbenzidine Dark purple stain
Alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitroblue Dark blue stain

tetrazolium
Chemiluminescent HRP Luminol/H2O2/p-iodophenol Blue light
Alkaline phosphatase 1,2-dioxetane derivatives Light
Alkaline phosphatase Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′- Light

(5′-chloro) tricyclo[3.3.1.13,7]decan} 4-yl)-1-phenyl
phosphate and derivatives (CSPD, CDP-Star)
For chromogenic detection, color develops directly on

The substrate used most often for chromogenic the membrane. The advantage of this type of detection is
detection is a mixture of nitroblue tetrazolium that the color can be observed and the reaction stopped
(NBT) and 5-bromo-4-chloro-3-indolyl phosphate at a time when there is an optimum signal-to-background
(BCIP). Upon dephosphorylation of BCIP by alka- ratio. In general, chromogenic detection is not as sensi-
line phosphatase, it is oxidized by NBT to give tive as chemiluminescent detection and can also result
a dark blue indigo dye as an oxidation product. in background noise, especially with probes labeled by
BCIP is reduced in the process and also yields a random priming.
blue product (see Fig. 5.19). The key to a successful blotting method is a high
signal-to-noise ratio. Ideally, the probe and detection
systems should yield a specific and robust signal. A high
As with radioactive detection, the chemiluminescent
specific signal, however, may be accompanied by back-
signal produced by the action of the enzyme on diox-
ground noise. Blocking agents are used with nonradio-
etane develops in the dark by autoradiography. The
active detection systems to avoid nonspecific binding of
release of light by phosphorylation of dioxetane occurs
conjugate to the membrane; however, blocking cannot
at the location on the membrane where the probe is
be so strong that it interferes with specific binding of
bound and darkens the light-sensitive film. Chemilumi-
the conjugate to the probe. Therefore, the specificity of
nescent detection can be stronger and develops faster
detection may be sacrificed to get a stronger signal. Con-
than radioactive detection. A disadvantage of earlier
versely, sensitivity may be sacrificed to generate a more
chemiluminescent detection was that it was harder to
specific signal.
control and sometimes produced nonspecific signals.
New substrates have been designed to minimize these
drawbacks. Unlike radioactive detection, in which INTERPRETATION OF RESULTS
testing the membrane with a Geiger counter can give an
indication of how “hot” the bands are and consequently Binding of a specific probe to its target immobilized on
how long to expose the membrane to the film, chemilu- a membrane results in the visualization of a “band” on
minescent detection may require developing films at dif- the membrane or film. A band is seen as a line running
ferent intervals to determine the optimum exposure time. across the width of the lane.
M 1 2 3 4 1 2 3 4
FIGURE 5.20 Example of a Southern blot result comparing FIGURE 5.21 Example of a northern or western blot result.
restriction fragment lengths of selected regions in different Lane 1 contains a positive control transcript or protein (arrow)
samples. Restriction digests of a genetic region can also show to verify the probe specificity and target size. Molecular-weight
differences in structure. The first lane (M) contains molecular- markers can also be used to estimate size as in Southern anal-
weight markers. Two samples with the same pattern (e.g., lanes ysis. The amount of gene product (expression level) is deter-
1 and 3) can be considered genetically similar. mined by the intensity of the signal from the test samples
relative to a control gene product (lower band in lanes 2 to 4).
The control transcript is used to correct for any differences in
Analysis of bands, that is, presence or absence or isolation or loading from sample to sample.
location in the lane, produced by Southern blot can be
straightforward or complex, depending on the sample
and the design of the procedure. Figure 5.20 is a depic-
internal standardized control (lower bands in all lanes)
tion of a Southern blot result. The bands shown can
ensures that a sample has low expression of target tran-
be visualized either on a membrane or on an autora-
script or protein product and that the low signal is not
diographic film, depending on the type of detection
due to technical difficulties.
system. If a gene locus has a known restriction pattern,
for instance, in lane 1, then samples can be tested to
compare their restriction patterns. In Figure 5.20, the
sample in lane 3 has the identical pattern; that is, both
ARRAY-BASED HYBRIDIZATION
lanes have the same number of bands, and the bands
Dot/Slot Blots
are all in the same location on the autoradiogram and
are likely to be very similar if not identical in sequence There are many variations on hybridization configura-
to the sample in lane 1. Southern blot cannot detect tions. In cases where the determination of the size of
tiny deletions or insertions of nucleotides or single- the target is not required, DNA and RNA can be more
nucleotide differences unless they affect a restriction quickly analyzed using dot blots or slot blots. These pro-
enzyme site. For some assays, cross-hybridization may cedures are applied to expression, mutation, and amplifi-
confuse results. These artifacts can be identified by their cation/deletion analyses.
presence in every lane at a constant size. For dot or slot blots, the target DNA or RNA is
Northern (or western) blots are used for the analysis deposited directly on the membrane by means of various
of gene expression, although they can also be used to devices, some with vacuum systems. A pipet can be used
analyze transcript size, transcript processing, and protein for procedures testing only a few samples. For dot blots,
modification. For these analyses, especially when esti- the target is deposited in a circle or dot. For slot blots,
mating expression, it is important to include an internal the target is deposited in an oblong bar (Fig. 5.22). Slot
control to correct for errors in isolation, gel loading, and blots are more accurate for quantification by densi-
transfer of samples. The amount of expression is then tometry scanning because they eliminate the error that
determined relative to the internal control (Fig. 5.21). may arise from scanning through a circular target. Dot
In the example shown, the target transcript or protein blots are useful for multiple qualitative analyses where
product (top bands in all lanes, indicated by the arrow) many targets are being compared, such as in mutational
is expressed in increasing amounts, left to right. The screening.
Macroarrays
In contrast to northern and Southern blots, dot (and slot)
blots offer the ability to test and analyze larger numbers
of samples at the same time. These methodologies are
limited, however, by the area of the substrate material,
the nitrocellulose membranes, and the volume of hybrid-
ization solution required to provide enough probe to
FIGURE 5.22 Example configuration of a dot blot (left) and produce an adequate signal for interpretation. In addi-
a slot blot (right). The target is spotted in duplicate, side by
tion, although up to several hundred test samples can
side, on the dot blot. The last two rows of spots contain posi-
tive, sensitivity, and negative control followed by a blank with be analyzed simultaneously on a dot blot, those samples
no target. The top two rows of the slot blot gel on the left can be tested for only one gene or gene product.
represent four samples spotted in duplicate, with positive, sen- A variation of this technique is the reverse dot blot,
sitivity, and negative control followed by a blank with no in which many different unlabeled probes are immobi-
target in the last four samples on the right. The bottom two lized on the membrane, and the test sample is labeled
rows represent a loading or normalization control that is often for hybridization with the immobilized probes. In this
useful in expression studies to confirm that equal amounts of configuration, the terminology can be confusing. The
DNA or RNA were spotted for each test sample. immobilized probe is now effectively the target, and
the labeled specimen DNA, RNA, or protein is actu-
ally the probe(s). Regardless of the designation, the
Dot and slot blots are performed most efficiently on
general idea is that a known sequence is immobilized at
less complex samples, such as cloned plasmids, PCR
a known location on the blot, and the amount of sample
products, or selected mRNA preparations. Without gel
that hybridizes to it is determined by the signal from the
resolution of the target fragments, it is important that
labeled sample.
the probe hybridization conditions be optimized because
Macroarrays are reverse dot blots of up to several
cross-hybridizations cannot be definitively distinguished
thousand targets on nitrocellulose membranes. Radio-
from true target identification. A negative control (DNA
active or chemiluminescent signals were typically used
of equal complexity but without the targeted sequence)
to detect hybridization of a labeled sample to the target
serves as the baseline for interpretation of these assays.
probes on the membrane. Macroarrays were created by
When performing expression analysis by slot or dot
spotting multiple probes onto nitrocellulose membranes.
blots, it is also important to include an amplification
The hybridization of labeled sample material was read
or normalization control, as shown on the slot blot in
by eye or with a phosphorimager (a quantitative imaging
Figure 5.22. This allows correction for loading or
device that uses storage phosphor technology instead of
sample differences. This control can also be analyzed on
x-ray film). For analysis, the signal intensity from test
a separate, duplicate membrane to avoid cross-reactions
“spots” was compared to control samples spotted on
between the test and control probes.
duplicate membranes.
Although macroarrays greatly increased the capac-
Genomic Array Technology ity to assess numerous targets, this system was limited
by the area of the membrane and the specimen require-
Array technology is a type of hybridization analysis
ments. As the target number increased, the volume of
allowing the simultaneous study of large numbers of
sample material required increased. This limits the utility
targets (or samples). Arrays are applied to gene (DNA)
of this method for small amounts of test material, espe-
amplification or deletion on comparative genome
cially as might be encountered with clinical specimens.
hybridization arrays and to gene-expression (RNA or
protein) analysis on expression arrays. There are several
Microarrays
approaches to array technology, including macroarrays,
microarrays, high-density oligonucleotide arrays, and In 1987, treated glass replaced nitrocellulose or nylon
microelectronic arrays. membranes for the production of arrays, increasing the
Solid Split Pin and Thermal Solenoid Piezoelectric

pin pin ring
FIGURE 5.23 Pen-type (left) and ink-jet (right) technologies used to spot arrays. Pen-type spotters physically contact the array
surface, in contrast to the ink-jet spotters, which do not.
versatility of array production. With improved spotting

technology and the ability to deposit very small target
spots on glass substrates, the macroarray evolved into
the microarray. Tens of thousands of targets could be
screened simultaneously in a very small area by min-
iaturizing the deposition of droplets (Fig. 5.23). Auto-
mated depositing systems (arrayers) can place more
than 80,000 spots on a glass substrate the size of a
microscope slide. The completion of the rough draft of FIGURE 5.24 A microarray, or DNA chip, is a glass slide
the human genome sequence revealed that the human carrying hundreds to thousands of probes. Arrays are some-
genome may consist of fewer than 30,000 genes. Thus, times supplied with fluorescent nucleotides for use in labeling
even with spotting representative sequences of each the test samples and software for identification of the probes
gene in triplicate, simultaneous screening of the entire bound with sample on the array by the array reader.
human genome on a single chip was within the scope of
array technology.
The larger nitrocellulose membrane of the macroarray,
then, was replaced by a glass microscope slide of the
microarray. The slide carrying the array of targets was
Advanced Concepts sometimes referred to as a chip (Fig. 5.24). This termi-
nology has led to some confusion of microarray tech-
The first automated arrayer was described in 1995 nology with lab-on-a-chip technology. Lab-on-a-chip is
by Patrick Brown at Stanford University.15 This a system of channels and reservoirs etched into glass or
and later versions of automated arrayers used other material where chemical reactions can take place.
pen-type contact to place a dot of probe material It has no relationship to microarray chips in this regard.
onto the substrate. Modifications of this technol- Array targets immobilized on the glass slide are
ogy include the incorporation of ink-jet printing usually DNA—either cDNAs, PCR products, or oligo-
systems to deposit specific targets at designated mers—however, targets can be DNA, RNA, or protein.
positions using thermal, solenoid, or piezoelectric Targets are spotted in triplicate and spaced across the
expulsion of the target material (see Fig. 5.23). chip to avoid any geographic artifacts that may occur
from uneven hybridization or other technical problems.
Mask Light
Activated
C A T A T
O O O OH OH O O A G C T G
T T T T T T C C T T C C G
DNA
Glass slide 10–25 nucleotides
FIGURE 5.25 Photolithographic target synthesis. A mask (left) allows light activation of the chip. When a nucleotide is added,
only the activated spots will covalently attach it (center right). The masking/activation process is repeated until the desired
sequences are generated at each position on the chip (right).
Test samples are usually cDNA-generated from sample positions on the chip. Proprietary photolithography
RNA but can, as well, be genomic DNA, RNA, or techniques allow for the highly efficient synthesis of
protein. short oligomers (10 to 25 bases long) on high-density
arrays (Fig. 5.25). These oligomers are then probed with
labeled fragments of the test sequences. Using this tech-
Advanced Concepts nology, hundreds of thousands of targets can be applied
to chips with high (single-base-pair) resolution. These
The study of the entire genome or sets of related types of high-density oligonucleotide arrays are used
genes is the field of genomics. When the combina- for mutation and polymorphism analysis, DNA methyla-
torial and interrelated functions of gene products tion analysis, and sequencing.
are known, observing the behavior of sets of genes Sample preparation for array analysis requires fluo-
or whole genomes is a more accurate method for rescent labeling of the test sample because microarrays
analyzing biological states or responses than iso- and other high-density arrays are read by automated flu-
lated studies of single genes. The complete set of orescent detection systems. The most frequent labeling
transcripts encoded by a genome is the transcrip- method used for RNA is the synthesis of cDNA or RNA
tome, and the set of encoded proteins is the pro- copies with the incorporation of labeled nucleotides. For
teome. One gene can encode multiple transcripts DNA, random priming or nick translation is used. Several
so that the transcriptome is more complex than alternative methods have also been developed.18,19
the genome. One transcript can give rise to more For gene-expression analyses, target probes immobi-
than one protein, making the proteome even more lized on the chips are hybridized with labeled mRNA
complex. Stanley Fields16 predicted that the pro- (cDNA) from treated cells or different cell types to
teome is likely to be 10 times more complex than assess the expression activity of the genes represented
the genome. The study of entire sets of proteins, on the chip. Arrays used for this application are classi-
or proteomics, is facilitated by array technology fied as expression arrays.20 Expression arrays measure
using antigen/antibody or receptor/ligand binding transcript or protein production relative to a reference
in the array format and mass spectrometry. control for each target gene isolated from untreated or
normal specimens (Fig. 5.26). For proteins, immobilized
antibodies (or protein ligands) are the probes, similar to
Another method used to deposit targets for array anal- their use in enzyme-linked immunoassays and western
ysis is DNA synthesis directly on the glass or silicon blots.
support.17 This technique uses sequence information to In contrast, comparative genome hybridization
design oligonucleotides and to selectively mask, acti- (array CGH) is designed to test DNA. This method is
vate, and covalently attach nucleotides at designated used to screen the genome or specific genomic loci for
Control Treated Control Treated Normal reference DNA
Test sample DNA
Single-color Dual-color
fluorescent fluorescent
labeling labeling
Hybridize Hybridize Microarray CGH
FIGURE 5.26 Samples are labeled, rather than probes, for

array analysis. At the left is single-color fluorescent labeling, Chromosome
where duplicate chips are hybridized separately and compared.
On the right is dual-color labeling, where test (treated) and ref-
erence (control) samples are labeled with different color fluors
and hybridized to the same chip. Locus
Cytogenetic location
21
deletions and amplifications. In this method, genomic FIGURE 5.27 Comparative genomic hybridization. Refer-
DNA is isolated, fragmented, and labeled for hybridiza- ence and test DNA are labeled with different fluors, repre-
tion on the chip (Fig. 5.27), which is analogous to the sented here as black and purple, respectively. After
cytogenetic technique done on metaphase chromosomes. hybridization, excess purple label indicates amplification of the
Array CGH can provide higher resolution and more test sample locus. Excess black label indicates deletion of the
defined genetic information than traditional cytogenetic test sample locus. Neutral or gray indicates equal test and ref-
analysis, but it is limited to the analysis of loci repre- erence DNA.
sented on the array. An advantage for clinical applica- been accumulated to determine the degree of nonspe-
tions is that genomic arrays can be performed on fixed cific binding and cross-hybridization that might occur
tissue and limiting samples. Methods have been devel- among and within a given set of sequences on an array.
oped to globally amplify whole genomes to enhance For instance, how much variation would result from
CGH analysis for limited samples, such as cell-free comparing two normal samples together multiple times?
DNA and circulating tumor cells.22,23 Background noise also affected the interpretation of
Reading microarrays requires a fluorescent reader and array results. Furthermore, as a result of passive hybrid-
analysis software. After correction for background noise ization, different sequences will have different binding
and normalization with standards, the software averages affinities under the same stringency conditions unless
signal intensity from duplicate or triplicate sample data. immobilized sequences are carefully designed to have
The results are reported as a relative amount of the ref- similar melting temperatures. For mutation analysis, the
erence and test signals. Depending on the program, vari- length of the immobilized probe is limited due to the use
ances of more than 2 to 3 standard deviations from 1 of a single hybridization condition for all sequences. For
(test = reference) are considered an indication of signifi- gene-expression applications, only relative, rather than
cant increases (test/reference greater than 1) or decreases absolute, quantification is possible.
(test/reference less than 1) in the test sample. These and other concerns have been addressed to
Several limitations of the array technology initially improve the reliability and consistency of array anal-
restrained the use of microarrays in the clinical labo- ysis. Baseline measurements, universal standards, and
ratory. The lack of established standards and controls recommended controls have been established. Arrays
for optimal binding prevented the calibration of arrays are seeing increased use in clinical laboratories due to
from one laboratory to another, and not enough data had improvements in price, applications, and availability of
instrumentation. Premade chips increase opportunities Labeled probe Single-stranded RNA

for medical applications, from targeted pathway analysis
to genome-wide studies. Minimal sample requirements
and comprehensive analysis with relatively small invest-
ments in time and labor are attractive features of array
technology.
Hybridization
Bead Array Technology

Analysis of multiple targets in a single specimen is the
key advantage of array technology. In microarrays, the
probes are immobilized on a solid support. The probes
may also be immobilized on beads, allowing hybridiza-
tion of the targets in the bead suspension. In this way, Nuclease
multiple suspensions can be tested simultaneously, for
example, in each well of a 96-well plate. In order to
distinguish specific probes carried on different beads,
the beads are color-coded with a particular shade of
red fluorescent dye. The sample is then labeled with a
green dye so that the combination of the target and bead
fluorescent signals indicates the presence or absence Nuclease – +
of a specific target. This technology is used for protein
and nucleic acid targets. Clinical tests using bead array Full-length probe
systems are available for infectious diseases and tissue
typing.
Target RNA hybrid

SOLUTION HYBRIDIZATION
FIGURE 5.28 Solution hybridization. Target RNAs are
In solution hybridization, neither the probe nor the hybridized to a labeled RNA or DNA carrying the comple-
target is immobilized. Probes and targets bind in solu- mentary sequence to the target. After digestion by a single-
tion, followed by resolution of the bound products. strand-specific nuclease, only the target:probe double-stranded
Solution hybridization has been used to measure hybrid remains. The hybrid can be visualized by the label on
mRNA expression, especially when there are low the probe after electrophoresis.
amounts of target RNA. One version of the method is
called RNase protection, or S1 mapping, for the S1 sin-
gle-strand–specific nuclease. In S1 mapping, the labeled northern blotting because no target can be lost during
probe is hybridized to the target sample in solution. electrophoresis and blotting. It is more applicable to
After digestion of excess probe by a single-strand–spe- RNA expression analysis due to limited sensitivity with
cific nuclease, the resulting labeled, double-stranded double-stranded DNA targets.
fragments are resolved by polyacrylamide gel electro- Another variation of solution hybridization is the
phoresis (Fig. 5.28). S1 mapping is useful for determin- capture of DNA probe:RNA target hybrids on a solid
ing the start point or termination point of transcripts. support or beads rather than by electrophoresis.25 For
Nuclease protection assays are also used to detect and these “sandwich”-type assays, two probes are used, both
quantify specific RNA targets from complex RNA mix- of which hybridize to the target RNA. One probe, the
tures. This technique is now performed using commer- capture probe, is biotinylated and will bind specifically
cial reagent sets.24 The procedure is more sensitive than to streptavidin immobilized on a plate or on magnetic
Increasing probe a. AGTCTGGGACGGCGCGGCAATCGCA

TCAGACCCTGCCGCGCCGTTAGCGT
Bound b. TCAAAAATCGAATATTTGCTTATCTA
AGTTTTTAGCTTATAAACGAATAGAT
Free
c. AGCTAAGCATCGAATTGGCCATCGTGTG
TCGATTCGTAGCTTAACCGGTAGCACAC
FIGURE 5.29 Gel mobility shift assay showing protein– d. CATCGCGATCTGCAATTACGACGATAA
protein or protein–DNA interaction. The labeled test substrate GTAGCGCTAGACGTTAATGCTGCTATT
is mixed with the probe in solution and then analyzed on a
polyacrylamide gel. If the test protein binds the probe protein 2. What is the purpose of denaturation of a double-
or DNA, the protein will shift up in the gel assay. stranded target DNA after electrophoresis and prior
to transfer in a Southern blot?
beads. The other probe, called the detection probe, is 3. Name two ways to permanently bind nucleic acid
detected by a monoclonal antibody directed against to nitrocellulose following transfer.
RNA:DNA hybrids or a covalently attached digoxigenin
molecule used to generate a chromogenic or chemilumi- 4. If a probe for a Southern blot is dissolved in a
nescent signal. hybridization buffer that contains 50% formamide,
Solution hybridization has also been applied to the is the stringency of hybridization higher or lower
analysis of protein–protein interactions and to nucleic than if there was no formamide?
acid–binding proteins using a gel mobility shift
assay.26,27 After mixing the labeled DNA or protein with 5. If a high concentration of NaCl was added to a
the test material, such as a cell lysate, a change in mobil- hybridization solution, how would the stringency
ity, usually a shift to slower migration, indicates binding be affected?
of a component in the test material to the probe protein
or nucleic acid (Fig. 5.29). This assay has been used to
6. Does an increase in temperature from 65°C to
identify trans factors that bind to cis-acting elements that
75°C during hybridization raise or lower the
control gene regulation. Solution hybridization can also
stringency?
be used to detect sequence changes in DNA or mutational
analysis. Hybridization methods offer the advantage of
7. At the end of the Southern blot procedure, what
direct analysis of nucleic acids at the sequence level
would the autoradiogram show if the stringency
without cloning of target sequences. The significance
was too high?
of hybridization methodology to clinical applications is
the direct discovery of molecular genetic information
from routine specimen types. Widely varying modi- 8. A northern blot is performed on an RNA
fications of the basic blotting methods have been and transcript with the sequence GUAGGUATGUA
will be developed for clinical and research applications. UUUGGGCGCGAACGCAAAA. The probe
Although amplification methods, specifically the PCR, sequence is GUAGGUATGUAUUUGGGCGCG.
have replaced many blotting procedures, some hybrid- Will this probe hybridize to the target
ization methods are still used in routine clinical analysis. transcript?
9. In an array CGH experiment, three test samples

were hybridized to three microarray chips. Each
STUDY QUESTIONS chip was spotted with eight gene probes (Genes
A–H). The following table shows the results of
1. Calculate the melting temperature of the following this assay expressed as the ratio of test DNA
DNA fragments using the sequences only: to reference DNA. Are any of the eight genes
consistently deleted or amplified in the test 8. Southern E. Detection of specific sequences among DNA frag-
samples? If so, which ones? ments separated by gel electrophoresis. Journal of Molecular
Biology 1975;98:503–517.
9. Bowen B, Steinberg J, Laemmli UK, Weintraub H. The detec-
Gene Sample 1 Sample 2 Sample 3 tion of DNA-binding proteins by protein blotting. Nucleic Acids
Research 1980;8:1–20.
A 1.06 0.99 1.01 10. Doessing H, Vester B. Locked and unlocked nucleosides in func-
tional nucleic acids. Molecules 2011;16:4511–4526.
B 0.45 0.55 0.43 11. Campbell M, Wengel J. Locked vs. unlocked nucleic acids (LNA
vs. UNA): contrasting structures work towards common therapeu-
C 1.01 1.05 1.06 tic goals. Chemical Society Reviews 2011;40:5680–5689.
12. Kohler G, Milstein C. Continuous cultures of fused cells secreting
D 0.98 1.00 0.97
antibody of predefined specificity. Nature 1975;256:495–497.
E 1.55 1.47 1.62 13. Giesen U, Kleider W, Berding C, Geiger A, Orum H, Nielsen PE.
A formula for thermal stability (Tm) prediction of PNA/DNA com-
F 0.98 1.06 1.01 plexes. Nucleic Acids Research 1998;21:5004–5006.
14. Britten RJ, Kohne DE. Repeated sequences in DNA. Science
G 1.00 0.99 0.99 1968;161:529–540.
15. Schena M, Shalon D, Davis RW, Brown PO. Quantitative mon-
H 1.08 1.09 0.90 itoring of gene expression patterns with a complementary DNA
microarray. Science 1995;270:467–470.
16. Fields S. Proteomics in genomeland. Science 2001;291:
1221–1224.
10. What are two differences between CGH arrays and 17. Lipschultz RJ, Fodor SP, Gingeras TR, Lockhart, DJ. High density
expression arrays? synthetic oligonucleotide arrays. Nature Genetics 1999;21:
20–24.
18. Maltas E, Malkondu S, Uyar P, Ozmen M. Fluorescent label-
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Chapter 6
Nucleic Acid Amplification
Outline Objectives
TARGET AMPLIFICATION 6.1 Compare and contrast the in vitro assays for
Polymerase Chain Reaction amplifying nucleic acids with regard to type of
Basic PCR Procedure target nucleic acid, principle, major elements of
Components of PCR the procedure, type of amplicon produced, major
Thermal Cyclers enzyme(s) employed, and applications.
The Reaction in PCR 6.2 Describe examples of modifications that have been
Control of PCR Contamination developed for PCR.
Mispriming 6.3 Discuss how amplicons are detected for each of the
PCR Product Cleanup amplification methods.
PCR Modifications 6.4 Design forward and reverse primers for a PCR, given
Transcription-Based Amplification Systems the target sequence.
Genomic Amplification Methods 6.5 Differentiate between target amplification and
Whole-Genome Amplification signal amplification.
Emulsion PCR
Surface Amplification (Bridge PCR)
Arbitrarily Primed PCR
PROBE AMPLIFICATION
Ligase Chain Reaction
Strand Displacement Amplification
Qβ Replicase
SIGNAL AMPLIFICATION
Branched DNA Amplification
Hybrid Capture Assays
Cleavage-Based Amplification
Cycling Probe
142
Chapter 6 • Nucleic Acid Amplification 143
Early analyses of nucleic acids were limited by the What Mullis had envisioned was the PCR. Over the
availability of DNA. Generating enough copies of a next months in the laboratory, he synthesized short oli-
single gene required propagation of millions of cells in gonucleotides (primers) complementary to sequences
culture or isolation of large amounts of genomic DNA. flanking a region of the human nerve growth factor and
If a gene had been cloned, many copies could be gener- tried to amplify the region from human DNA, but the
ated on bacterial plasmids, but this preparation was labo- experiment did not work. Not sure of the nucleotide
rious, and some sequences were resistant to propagation sequence information he had on the human gene, he
in this manner. tried a more defined target. The first successful ampli-
The ability to amplify a specific DNA sequence fication was a short fragment of the Escherichia coli
opened the possibility of analyzing at the nucleotide level plasmid, pBR322. The first paper describing a practical
virtually any piece of DNA in nature. The first specific application, the amplification of beta-globin and anal-
amplification method of any type was the polymerase ysis for diagnosis of patients with sickle cell anemia,
chain reaction (PCR). Other amplification methods was published 2 years later.3 He called the method a
have been developed based on making modifications to “polymerase-catalyzed chain reaction” because DNA
PCR. The methods that have been developed to amplify polymerase was the enzyme he used to drive the rep-
nucleic acids can be divided into three groups, based on lication of DNA in a chain reaction. The name was
whether the target nucleic acid itself, a probe specific quickly shortened to PCR. Since PCR was first per-
for the target sequence, or the signal used to detect the formed, it has become increasingly user-friendly, more
target nucleic acid is amplified. These methods are dis- automated, and more amenable to use in a clinical
cussed in this chapter. laboratory.
Basic PCR Procedure

TARGET AMPLIFICATION DNA replication in a cell requires an existing
double-stranded DNA as the template to give the order
Target amplification involves making many copies of
of the nucleotide bases; the deoxyribonucleotide bases
a specific DNA sequence. This is analogous to growing
in the form of deoxynucleotide triphosphates (dNTPs)—
cells in culture and allowing the cells to replicate their
dATP, dGTP, dCTP, dTTP; DNA polymerase to catalyze
nucleic acid as well as themselves so that, for example,
the addition of nucleotides to the growing strand; and a
they can be visualized on an agar plate. Waiting for cells
primer (synthesized by primase in vivo) to which DNA
to replicate to detectable levels can take days to weeks
polymerase adds subsequent bases.
or months, whereas replicating the nucleic acid in vitro
PCR essentially duplicates the in vivo replication of
only takes minutes to hours. PCR is the first and proto-
DNA in vitro, using the same components (Table 6.1) to
typical method for amplifying target nucleic acid.
replicate DNA, with the same end result: one copy of
double-stranded DNA becoming two copies (Fig. 6.1). In
less than 2 hours, PCR can produce millions of copies,
Polymerase Chain Reaction
collectively called amplicons, of DNA in contrast to
Kary Mullis visualized the idea of amplifying DNA in days for a cell to produce the same number of copies in
vitro in 1983 while driving one night on a California vivo. The real advantage of PCR is the ability to amplify
highway.1,2 In the process of working through a mutation- specific targets. Just as the Southern blot first allowed
detection method, Mullis realized that by adding all four analysis of specific regions in a complex background,
nucleotides to his reaction mix, he could double his test PCR presents the opportunity to amplify and effectively
target, a short region of double-stranded DNA, giving clone the target sequences. The amplified target can then
him 21, or 2, copies. If he repeated the process, the target be subjected to innumerable analytical procedures.
would double again, giving 22, or 4, copies. After N dou- To perform a PCR amplification, the components
blings, he would have 2N copies of his target. If N = 30 of the reaction—DNA template, short oligonucleotide
or 40, there would be millions of copies. primers, nucleotides, polymerase, and buffers—are
subjected to an amplification program, which consists

TABLE 6.1 Components of a Typical Reaction of a specified number of cycles that are divided into
in PCR steps during which the samples are held at particular
temperatures for designated times. Table 6.2 shows the
Component Purpose steps of a common three-step PCR cycle.
0.25 mM each primer Directs DNA synthesis to The amplification program starts with one double-
(oligodeoxynucleotides) the desired region stranded DNA target. In the first step (denaturation),
the double-stranded DNA is denatured into two single
0.2 mM each dATP, dCTP, dGTP, Building blocks that strands (Fig. 6.2). This is accomplished by heating the
dTTP extend the primers
sample at 94°C to 96°C for several seconds to several
50 mM KCl Monovalent cation (salt), minutes, depending on the template. The initial dena-
for optimal hybridization turation step is lengthened for genomic or other large
of primers to template DNA template fragments. Subsequent denaturations can
10 mM Tris, pH 8.4 Buffer to maintain be shorter.
optimal pH for the
enzyme reaction
1.5 mM MgCl2 Divalent cation, required TABLE 6.2 Elements of a PCR Cycle
by the enzyme
Step Temperature (°C) Time (sec)
2.5 units polymerase The polymerase enzyme
that extends the primers Denaturation 90–96 20–60
(adds dNTPs)
Annealing 50–70 20–90
102–105 copies of template Sample DNA that is being
tested Extension 68–75 10–60
Region under
investigation
Template DNA
5′ 3′
G A A T CG T CG A GC T GC T A GC T T T G T T CG A
GA A A C A A
Forward Reverse
A T CG T C
C T T A GC A GC T CG A CG A T CG A A A C A A GC T
3′ 5′
Template DNA
PCR
5′ 3′ FIGURE 6.1 The components and result of

A T CG T CG A GC T GC T A GC T T T G T T
T A GC A GC T CG A CG A T CGA A A C A A a PCR. Oligodeoxynucleotides (primers) are
designed to hybridize to sequences flanking the
DNA region under investigation. The poly-
A T CG T CG A GC T GC T A GC T T T G T T merase extends the forward and reverse primers
T A GC A GC T CG A CG A T CG A A A C A A making many copies of the region flanked by
3′ 5′ the primer sequences, the PCR product.
5′ then heat the reaction to denature the dNTP-

Primers
extended oligos and add an excess of unextended
oligos and the ddNTPs. As he further considered
the modification to his method, he realized that if
DNA
the extension of an oligo in the preliminary reac-
Region to be
3′ tion crossed the point where the other oligo was
amplified
5′ bound on the opposite strand, he would make a
new copy of the region between the oligos. He
considered the new copy an additional advantage
3′
because it would improve the sensitivity of this
FIGURE 6.2 Denaturation of the DNA target. The region to method by doubling the target. Then, he thought,
be amplified is shown in dark purple. The primers (black) are what if he did it again? The target would double
present in vast excess. again. If he added dNTPs intentionally, he could
do it over and over again, doubling each time and
making 2N copies, where N is the number of times
the region is replicated.
In his own words: “I stopped the car at mile
Histooricaal Higghlligghtts marker 46,7 on Highway 128. In the glove com-
partment I found some paper and a pen. I con-
Kary Mullis was working at Cetus Corporation, firmed that two to the tenth power was about a
where he synthesized short, single-stranded DNA thousand and that two to the twentieth power was
molecules, or oligodeoxynucleotides (oligos), about a million and that two to the thirtieth power
used by other laboratories. Mullis also tinkered was around a billion, close to the number of base
with the oligos he made. One night, as he drove pairs in the human genome. Once I had cycled
through the mountains of northern California, this reaction thirty times I would be able to repro-
Mullis thought about a method he had designed duce the sequence of a sample with an immense
to detect mutations in DNA. His scheme was signal and almost no background.”2
to add radioactive dideoxynucleotides—ddATP,
ddCTP, ddGTP, ddTTP—to four separate DNA
synthesis reactions containing oligos, the test
DNA template, and polymerase. In each reac- The next step in the amplification program, most criti-
tion, the complementary oligo would hybridize cal for the specificity of the PCR, is the annealing step.
to the template, and the polymerase would extend This is the second step of the PCR cycle where the two
the oligo with the dideoxynucleotide—but only oligonucleotide primers that will prime the synthesis of
the dideoxynucleotide that was complementary DNA anneal (hybridize) to complementary sequences
to the next nucleotide in the template. He could on the template (Fig. 6.3). The primers dictate the part
then determine in which of the four tubes the of the template that will be amplified; in other words,
oligo was extended with a radioactive ddNTP by the primers determine the specificity of the amplifica-
gel electrophoresis. tion. It is important that the annealing temperature be
He thought he might improve the method by optimized with the primers and reaction conditions.
using a double-stranded template and priming Annealing temperatures will range from 50°C to 70°C
synthesis on both strands, instead of one at a and are usually established empirically. A starting point
time. Because the results of the synthesis reaction can be determined using the Tm of the primer sequences.
would be affected by contaminating deoxynucle- Reaction conditions, salt concentration, primer sequence
otides (dNTPs) in the reagent mix, Mullis consid- mismatches, template condition, and secondary struc-
ered running a preliminary reaction without the ture will all affect the real Tm of the primers in the
ddNTPs to use up any dNTPs present. He would reaction.
DNA DNA
Region of interest 3′
5′ 3′ 5′
Primer
Primer
3′ 5′
3′ 5′
FIGURE 6.3 In the second step of the PCR cycle, annealing,

the primers hybridize to their complementary sequences on FIGURE 6.4 DNA polymerase catalyzes the addition of
each strand of the denatured template. The primers are designed deoxynucleotide triphosphates (dNTPs) to the primers, using
to hybridize to the sequences flanking the region of interest. the sample DNA as the template. This completes one PCR
cycle. In the original template, there was one copy of the target
region. Now, after one cycle, there are two copies.
Histooricaal Higghlligghtts road. In about a mile it occurred to me that the

Mullis’s original method, using ddNTPs and oligonucleotides could be placed at some arbi-
oligos to detect mutations, is still in use today. trary distance from each other, not just flanking
For example, fluorescent polarization–template- a base pair and that I could make an arbitrarily
directed dye terminator incorporation uses fluo- large number of copies of any sequence I chose
rescently labeled ddNTPs to distinguish which and what’s more, most of the copies after a few
ddNTP is added to the oligo. Another extension/ cycles would be the same size. That size would
termination assay, Homogeneous MassExtend, be up to me. They would look like restriction
is a similar method, using mass spectrometry to fragments on a gel. I stopped the car again. Dear
analyze the extension products. Both of these Thor!, I exclaimed. I had solved the most annoy-
methods were used in the Human Haplotype ing problems in DNA chemistry in a single light-
Mapping (HapMap) Project, which mapped mil- ning bolt. Abundance and distinction. With two
lions of single-nucleotide differences in human oligonucleotides, DNA polymerase, and the four
DNA populations. nucleoside triphosphates I could make as much of
a DNA sequence as I wanted and I could make it
on a fragment of a specific size that I could dis-
The third and last step of the PCR cycle is the prim- tinguish easily.”2
er-extension step (Fig. 6.4). This is where DNA syn-
thesis occurs. In this step, the polymerase synthesizes
a copy of the template DNA by adding nucleotides to
At the end of the three steps, or one cycle (denaturation,
the hybridized primers. DNA polymerase replicates the
primer annealing, and primer extension), one copy of
template DNA by simultaneously extending the primers
double-stranded DNA has been replicated into two dou-
on both strands of the template. This step occurs at the
ble-stranded copies. Returning to the denaturing tem-
optimal temperature of the enzyme, 68°C to 72°C.
perature starts the second cycle (Fig. 6.5), with the end
result being a doubling in the number of double-stranded
Histooricaal Higghlligghtts DNA molecules again. At the end of the PCR program,
millions of copies of the original region defined by the
As Kary Mullis realized early on, the key to the primer sequences will have been generated. Following
brilliance of PCR is that primers can be designed is a more detailed discussion of each of the components
to target specific sequences: “I drove on down the of PCR.
Target region Primers

The primers are the critical component of the reac-
tion because they determine the specificity of the
PCR. Primers are analogous to the probes in blot-
ting and hybridization procedures. Primers are chem-
ically manufactured on a DNA synthesizer. Most
laboratories purchase primers from commercial pro-
viders. Primer sequences are submitted in text form,
along with other specifications, such as amount, degree
of purification, and any modifications, such as biotin or
fluorescent dyes.
Primers are designed to contain sequences comple-
mentary to sites flanking the region to be analyzed.
Primer design is therefore a critical aspect of the PCR.
Primers are single-stranded DNA fragments, usually 20
to 30 bases in length. The forward primer hybridizes to
the complementary strand just 5′ to the sequences to be
FIGURE 6.5 The products of the first cycle are again repli- amplified. The reverse primer hybridizes just 3′ to the
cated in the second PCR cycle, yielding four copies of the sequence to be amplified (see Fig. 6.1). Thus, the design
target region. At each cycle the number of copies of the target of primers requires some knowledge of the target region.
region doubles. In an ideal PCR, the PCR product (amplicon) The placement of the primers will also dictate the size of
is composed of 2N copies of the target region, where the amplified product.
N = number of PCR cycles.
The nucleotide sequence orders of forward and
reverse primers are designed using genomic sequences
available from the National Center for Biological Infor-
mation or other resources. When procedures are pub-
Advanced Concepts lished, the primer sequences are provided in the methods
descriptions. Primers can be designed manually, or
In some cases, the primer annealing temperature alternatively, primer sequences can be automatically
is close enough to the extension temperature that generated by software programs from an input target
the reaction can proceed with only two tempera- sequence and chosen parameters, such as desired ampl-
ture changes. This is two-step PCR, as opposed to icon length.
three-step PCR that requires a different tempera- Hybridization of primers is subject to the same
ture for all three steps. physical limitations as probe hybridization. The primer
sequence (% GC) and length affect the conditions in
which the primer will bind to its target. The approxi-
mate melting temperature, or Tm, of the primers can be
Components of PCR
calculated using the equation for short DNA fragments
PCR is a method of in vitro DNA synthesis. Therefore, described in Chapter 5. This primer Tm then serves as a
to perform PCR, all of the components necessary for the starting point for setting the optimal annealing tempera-
replication of DNA in vivo are combined in optimal con- ture, the critical step for the specificity of the amplifica-
centrations for replication of DNA to occur in vitro. This tion reaction. Primers should be designed such that the
includes the template to be copied; primers to prime syn- forward and reverse primers have similar Tm so that both
thesis of the template; nucleotides; polymerase enzyme; will hybridize optimally at the same annealing tempera-
and buffer components. including monovalent and diva- ture. Tm can be adjusted by increasing the length of the
lent cations. to provide optimal conditions for accurate primers or by placing the primers in areas with more or
and efficient replication. fewer Gs and Cs in the template.
Intended target Unintended

sequence sequence
5′ 3′
3′ 5′
Primer
misprime
5′ 3′
3′ 5′
FIGURE 6.6 Mispriming of one primer
creates an unintended product that could
interfere with subsequent interpretation. Mis-
priming can also occur in regions unrelated to
the intended target sequence.
mispriming will carry the primer sequence and become

Advanced Concepts a template for subsequent cycles of amplification
(Fig. 6.6). Eventually, misprimed products will take
Most laboratories purchase primers from a man- components away from the intended reaction. They may
ufacturer by submitting the required sequences, also interfere with proper interpretation of results or
amount required (scale of synthesis), and level with ensuing procedures, such as sequencing or muta-
of purification. Standard primer orders are on tion analysis. Secondary structure (internal folding and
the 50-to-200-nm scale of synthesis. Higher hybridization within DNA strands) may also interfere
amounts (1 to 50 μM) are more expensive to pur- with PCR. Primer sequences that have internal homol-
chase per base. Primers are synthesized from an ogies, especially at the 3′ end, or homologies with the
immobilized 3′ end to the 5′ end so that incom- other member of the primer pair may not work as well
plete primer molecules will be shortened from the in the PCR. An artifact often observed in the PCR is the
5′ end. Purification of the synthesized primers may occurrence of primer dimers, which are PCR products
be performed by cartridge or column binding and that are approximately double the size of the primers.
washing, high-performance liquid chromatogra- They result from the binding of primers onto each other
phy (HPLC), or polyacrylamide gel electropho- through short (2- to 3-base) homologies at their 3′ ends
resis (PAGE). HPLC and PAGE purification can and the copying of each primer sequence (Fig. 6.7). The
remove incomplete synthesis products. Primers resulting doublet is then a very efficient target for subse-
may also be labeled at the time of synthesis with quent amplification.
fluorescent dyes, thiolation, biotinylation, or other The entire primer sequence does not have to bind to
modifiers. the template to prime synthesis; however, the 3′ nucleo-
tide position is critical for extension of the primer. The
polymerase will not form a phosphodiester bond if the
Primer sequence determines the accuracy of binding to 3′ end of the primer is not hydrogen-bonded to the tem-
its complementary sequence and not to other sequences. plate. This characteristic of primer binding has been
Just as cross-hybridization can occur with blot hybrid- exploited to modify the PCR procedure for mutation
ization, aberrant primer binding, or mispriming, can analysis of the template. There is no such strict require-
occur in PCR. A fragment synthesized as a result of ment for complementarity on the 5′ end of the primer.
5′ 3′ 5′ 3′
3′ 5′
3′ 5′ Primer
Any sequence
3′ 5′
5′ 3′
3′ 5′
5′ 3′
PCR product with
any sequence attached
3′ 5′ FIGURE 6.8 Sequences unrelated to the template can be
FIGURE 6.7 Formation of primer dimers occurs when there added to the 5′ end of the primer. After PCR, the sequence will
are three or more complementary bases at the 3′ end of the be on the end of the PCR product. These tailed primers can
primers. With the primers in excess, these will hybridize during add useful sequences to one, as shown, or both ends of the
the annealing step (vertical lines), and the primers will be PCR product. The 5′ end of the primer can also carry non-DNA
extended by the polymerase (dotted line) using the opposite molecules, such as fluorescent labels for detection of the
primer as the template. The resulting product, denatured in the product in capillary electrophoresis or biotin for capture of the
next cycle, will compete for primers with the intended PCR product.
template.
nucleotide bases. Templates with high GC content and
secondary structure may prove more difficult to optimize
Noncomplementary extensions or tails can be added to
for amplification.
the 5′ end of the primer sequences to introduce useful
additions to the final PCR product, such as restriction
enzyme sites, promoters, or binding sites for other Advanced Concepts
primers. These tailed primers are designed to add or
alter sequences to one or both ends of the PCR product Reagent systems have been designed to facilitate
(Fig. 6.8). amplification of targets with high GC content.
DNA Template These systems incorporate an analog of dGTP,
In a clinical sample, depending on the application, the deaza dGTP, to destabilize secondary structure
template may be derived from the patient’s genomic or formed by G:C base pairing. Deaza dGTP inter-
mitochondrial DNA or from viruses, bacteria, fungi, or feres with EtBr staining in gels and is best used in
parasites that might be infecting the patient. Genomic procedures with other types of detection, such as
DNA will have only one or two copies per cell equiv- autoradiography.
alent of single-copy genes to serve as amplification
targets. With robust PCR reagents and conditions, nano-
gram amounts of genomic DNA are sufficient for consis- Deoxyribonucleotide Bases
tent results. For routine clinical analysis, 100 ng to 1 μg An equimolar mixture of the four deoxynucleotide tri-
of DNA is usually used. Lesser amounts are required phosphates (dNTPs)—adenine, thymine, guanine, and
for more defined template preparations, such as cloned cytosine—is added to the synthesis reaction in concen-
target DNA or product from a previous amplification. trations sufficient to support the exponential increase
The best templates are in good condition, free of con- of copies of the template. Standard procedures require
taminating proteins, and without nicks or breaks that 0.1 to 0.5 mM concentrations of each nucleotide. Substi-
can stop DNA synthesis or cause mis-incorporation of tuted or labeled nucleotides such as deaza dGTP may be
included in the reaction for special applications. These procedure and would maintain its activity throughout
nucleotides will require empirical optimization for best the heating and cooling cycles. Other enzymes, such as
results. Tth polymerase, from Thermus thermophilus, were sub-
The concentration and purity of dNTP preparations sequently exploited for laboratory use. Tth polymerase
affect the efficiency of the PCR reaction. A single also has reverse-transcriptase activity, so it can be used
solution containing a mixture of all four nucleotides is in reverse-transcriptase PCR (RT-PCR) in which the
most convenient and lowers pipetting errors compared starting material is an RNA template. The addition of
with four separate nucleotide solutions. The four dNTP proofreading enzymes, for example, Vent polymerase,
working concentrations should be higher than the esti- allows Taq or Tth polymerase to generate large products
mated Km of each dNTP (10 to 15 mM, the concen- over 30,000 bases in length.
tration of substrate at half maximal enzyme velocity). The nomenclature for the polymerases is derived
Higher concentrations (at least 10 to 100 mM) are rec- from the organism from which each enzyme comes,
ommended for storage because storing dNTPs in lower similar to the nomenclature for restriction enzymes.
concentrations results in hydrolysis to dNDP and dNMP. For example, for the Taq polymerase, the T comes from
Nucleotide di- and monophosphates can also result from the genus name, Thermus, and the aq comes from the
poor manufacturing conditions or contamination with species name, aquaticus.
heavy metals. These molecules will inhibit the PCR Cloning of the genes coding for these polymerases
reaction. has led to modified versions of the polymerase
enzymes, such as the Stoffel fragment lacking the 289
Histooricaal Higghlligghtts N-terminal amino acids of Taq polymerase and its
inherent 3′ to 5′ exonuclease activity.5 The half-life of
The first “Molecule of the Year” selected by the Stoffel fragment at high temperatures is about twice
Science magazine was Taq DNA polymerase.4 that of Taq polymerase, and it has a broader range of
Development of the heat-stable enzyme is one optimal MgCl2 concentrations (2 to 10 mM) than Taq.
factor responsible for the vast increase in the use This enzyme is recommended for allele-specific PCR
of PCR from its first reported use in 1985.3 The and for amplification of regions with high GC content.
ability to continuously cycle through the heat Further modified versions of the Taq enzymes retaining
steps of the PCR without loss of enzyme activity 3′ to 5′ exonuclease, but not 5′ to 3′ exonuclease activ-
was a major advance in PCR technology. ity, are used where high fidelity (accurate copying of
the template) is important. Other variants of Taq poly-
merase, ThermoSequenase and T7 Sequenase, efficiently
DNA Polymerase incorporate dideoxy nucleoside triphosphates (NTPs)
Automation of the PCR procedure was greatly facili- for application to chain termination sequencing. A trun-
tated by the discovery of the thermostable enzyme Taq cated version of Taq polymerase has mutations render-
polymerase. When Kary Mullis first performed PCR, he ing it resistant to inhibitors present in whole blood, a
used the DNA polymerase isolated from E. coli. With characteristic applicable to clinical analysis.
every denaturation step, however, the high temperature Despite its thermal stability, the Taq polymerase
inactivated the enzyme. Thus, after each round of dena- enzyme is still subject to loss of activity under adverse
turation, additional E. coli DNA polymerase had to be conditions. For example, although mixing is important
added to the tube. This was labor-intensive, lowered for buffers and other solutions, especially after thawing,
reaction efficiency, and provided additional opportuni- vigorous agitation of the polymerase enzymes is not rec-
ties for the introduction of contaminants into the reac- ommended. This can result in mechanical shearing, alter-
tion tube. ing of the secondary and tertiary structures of complex
Taq polymerase was isolated from the thermophilic enzymes like the polymerases, and irreversible denatur-
bacterium Thermus aquaticus. Using an enzyme derived ation. Mixing also introduces air to the liquid, infusing
from a thermophilic bacterium meant that the DNA the air–liquid interface with bubbles, which can also
polymerase could be added once at the beginning of the cause damage. Most of the enzymes used in molecular
biology have complex tertiary structures (with multiple increasing the availability for primer binding. Chaotropic
subunits) and will lose enzyme activity upon vigorous agents (detergents), such as Triton X-100, glycerol, and
mixing. In contrast, tiny proteins like RNase can rena- dimethyl sulfoxide, added at concentrations of 1% to
ture and are therefore resistant to this type of treatment. 10% may also reduce secondary structure to allow poly-
merase extension through difficult areas. These agents
PCR Buffers contribute to the stability of the enzyme as well.
PCR buffers provide the optimal conditions for enzyme Enzymes are usually supplied with buffers optimized
activity. Potassium chloride (20 to 100 mM), ammonium by the manufacturer. Commercial PCR buffer enhancers
sulfate (15 to 30 mM), or other sources of monovalent of proprietary composition may also be purchased to
cations are important buffer components. These salts optimize difficult reactions. Often, the buffer and its
affect the denaturing and annealing temperatures of the ingredients are mixed with the nucleotide bases and
DNA and the enzyme activity. An increase in salt con- stored as aliquots of a master mix. The enzyme, target,
centration makes longer DNA products denature more and primers are then added when necessary. Dedicated
slowly than shorter DNA products during the amplifica- master mixes will also include the primers, so only the
tion process, so shorter molecules will be amplified pref- target sequences must be added to these mixes.
erentially. The influence of buffer/salt conditions varies
with different primers and templates.
Thermal Cyclers
Divalent cations, provided by magnesium chloride,
also affect primer annealing and are very important for The first PCRs were performed using multiple water
enzyme activity. Magnesium requirements will vary baths or heat blocks set at the required temperatures for
depending on other components of the reaction mix; for each of the steps of the PCR cycle. The tubes were man-
example, each NTP will take up one magnesium atom. ually moved from one temperature to another. In addi-
Furthermore, the presence of ethylenediaminetetraacetic tion, before the discovery of thermostable enzymes, new
acid (EDTA) or other chelators will lower the amount of enzyme had to be added after each denaturation step,
magnesium available for the enzyme. Too few Mg2+ ions further slowing the procedure and increasing the chance
will lower enzyme efficiency, resulting in a low yield of of error and contamination.
PCR product. Overly high Mg2+ concentrations promote Automation of this tedious process was greatly facil-
misincorporation and thus increase the yield of nonspe- itated by the availability of the heat-stable enzymes.
cific products. Lower Mg2+ concentrations are desirable To accomplish the PCR, then, an instrument must only
when the fidelity of the PCR is critical. The recom- manage temperature according to a scheduled amplifica-
mended range of MgCl2 concentration is 1 to 4 mM tion program. Thermal cyclers, or thermocyclers, were
in standard reaction conditions. If the DNA samples thus designed to rapidly and automatically ramp (change)
contain EDTA or other chelators, the MgCl2 concentra- to the required incubation temperatures, holding at each
tion in the reaction mixture should be adjusted accord- one for designated periods.
ingly. Tris buffer and additional buffer components are Early versions of thermal cyclers were designed as
also important for optimal enzyme activity and accurate heater/coolers with programmable memory to record the
amplification of the intended product; 10 mM Tris-HCl appropriate reaction conditions. Compared with modern
maintains the proper pH of the buffer, usually between models, the memory available for recording the reaction
pH 8 and pH 9.5. As with other PCR components, the conditions and sample was limited. Wax or oil (vapor
optimal conditions are established empirically. barriers) had to be added to the reactions to prevent
Accessory components are sometimes used to opti- condensation of the sample on the tops of the tubes
mize reactions. Bovine serum albumin (10 to 100 μg/ during the temperature changes. The layer of wax or oil
mL) binds inhibitors and stabilizes the enzyme. Dithioth- made subsequent sample handling more difficult. Later,
reitol (0.01 mM) provides reducing conditions that may thermal cycler models were designed with heated lids
enhance enzyme activity. Formamide (1% to 10%) added that eliminated the requirement for vapor barriers.
to the reaction mixture will lower the denaturing tem- The several available versions of thermal cyclers
perature of DNA with high secondary structure, thereby differ in heating and/or refrigeration systems as well as
the programmable software within the units. Some hold Molecular-weight Reagent blank
samples in open chambers for air heating and cooling; markers
others hold samples in blocks designed to accommodate
0.2-mL tubes, usually in a 96-well format. Some models
have interchangeable blocks to accommodate amplifica-
tion in different sizes and numbers of tubes or in situ
amplification of nucleic acid targets in tissue on slides.
A cycler may run more than one block independently
so that different PCR programs can be performed simul-
taneously. Rapid PCR systems work with small sample
volumes in chambers that can be heated and cooled
quickly by changing the air or specially designed block
temperature surrounding the samples.6 Real-time PCR
systems are equipped with fluorescent detectors to (Misprime)
measure the PCR product as the reaction proceeds. PCR
can also be performed in a microchip device in which
1- to 2-μL samples are forced through tiny channels
PCR
etched in a glass chip, passing through temperature product
zones as the chip rests on a specially adapted heat block
or microwave heating system.7,8
(Primer
dimers)
The Reaction in PCR
For routine PCR, an appropriate amount of DNA that
has been isolated from a test specimen is mixed with the
other PCR components, either separately or as a master FIGURE 6.9 Example of PCR products after resolution on an
mix. The final volume of the reaction mix varies from agarose gel and staining with ethidium bromide. Molecu-
1 to 50 μL for most PCR procedures. Thermal cyclers lar-weight markers in the first gel lane are used to estimate the
size of the PCR product. The intended product is 125 bp. Arti-
take thin-walled tubes, tube strips, or 96-well or 384-
factual primer dimers and misprimed products are also present.
well plates with 0.04- to 0.2-mL volume capacity. Absence of products in the last gel lane (reagent blank) con-
Preparation of the specimen for PCR is classified as a firms that there is no contamination in the master mix.
pre-PCR procedure. To avoid contamination (see fol-
lowing discussion), it is recommended that the pre-PCR
is analyzed by gel or capillary electrophoresis. Depend-
work be performed in a designated area that is clean and
ing on the application, the size, presence, or intensity
free of amplified products or other extraneous DNA. The
of PCR products is observed after electrophoresis. An
sample tubes are then loaded into the thermal cycler pro-
example of the results from a PCR run is shown in
grammed with the temperatures and times for each step
Figure 6.9.
of the PCR cycle, the number of cycles to be completed
(usually 30 to 50), the conditions for ramping from step Controls for PCR
to step, and the temperature at which to hold the tubes As with any diagnostic assay, running the correct con-
once all of the cycles are complete. Because the PCR trols during every PCR run is essential for ensuring
products are stable at the holding temperature, the tech- and maintaining the accuracy of the assay. Positive
nologist does not have to retrieve the samples from the controls ensure that the enzyme is active, the buffer is
thermal cycler immediately after the PCR program is optimal, the primers are priming the right sequences,
concluded. and the thermal cycler is cycling appropriately. A nega-
After PCR, a variety of methods can be used to tive control without DNA (also called a contamination
analyze the product. Most commonly, the PCR product control or reagent blank) ensures that the reaction mix
is not contaminated with template DNA or amplified when tubes are uncapped and when the amplified DNA
products from a previous run. A negative control with is pipetted. This PCR product is a perfect template for
DNA that lacks the target sequence (negative template primer binding and amplification in a subsequent PCR
control) ensures that the primers are not annealing to using the same primers. Contamination control proce-
nontarget sequences of DNA. In some applications of dures, therefore, are mainly directed toward eliminating
PCR, an internal amplification control is included that PCR product from the setup reaction.
contains a second set of primers and an unrelated target Contamination is controlled both physically and
added to the reaction mix. Alternatively, the amplifica- chemically. Physically, the best way to avoid PCR car-
tion control can be a synthetic template containing the ryover is to physically separate the pre-PCR areas from
target primer-binding sites but producing a larger product the post-PCR analysis areas. Positive airflow, airlocks,
than the target analyte. These controls demonstrate that and more extensive measures are taken by high-through-
the reaction is working even if the test sample is not put laboratories that process large numbers of samples
amplified. Amplification controls are amplified prefer- and test for a limited number of amplification targets.
ably in the same tube with the test reaction, although Most laboratories can separate these areas by assigning
it is acceptable to perform the amplification control in separate rooms or using isolation cabinets. Equipment,
a duplicate reaction mix if the control cannot easily be including laboratory gowns and gloves, and reagents
distinguished from the target product in the same reac- should be dedicated to either pre- or post-PCR. Gloves
tion. This type of control is most important when PCR should be changed if contaminated areas are touched
results are reported as positive or negative, “negative” before proceeding with the PCR setup. Items can flow
meaning that the target sequences are not present. The from the pre- to the post-PCR area but not in the oppo-
amplification control is critical to distinguish between a site direction, without decontamination.
true negative for the sample and an amplification failure Ultraviolet (UV) light has been used to decontaminate
(false negative). and maintain pre-PCR areas.9 UV light catalyzes single-
and double-strand breaks in the DNA that will then inter-
fere with replication. Isolation cabinets are equipped
Control of PCR Contamination
with UV light sources that are turned on for about
Contamination is a significant concern in “open-tube” 20 minutes after the box has been used. The effectiveness
methods that involve target amplification and manipula- of UV light may be increased by the addition of pso-
tion of the PCR product. The nature of the amplification ralens to amplification products after analysis. Psoralens
procedure is such that, theoretically, a single molecule intercalate between the bases of double-stranded DNA,
will give rise to a product. This is of great concern in and in the presence of long-wave UV light, they cova-
the medical or forensic laboratory where results may lently attach to the thymidines, uracils, and cytidines in
be interpreted based on the presence, absence, size, or the DNA chain. The bulky adducts of the psoralens pre-
amount of a PCR product. With modern reagent systems vent denaturation and amplification of the treated DNA.
designed for robust amplification of challenging spec- Although convenient and effective for antimicro-
imens, such as paraffin-embedded tissues or samples bial decontamination,10 UV treatment may not be the
with low cell numbers, the balance between aggressive most effective decontaminant for nucleic acid contam-
amplification of the intended target and avoidance of a ination.11-13 The efficiency of UV-light treatment for
contaminating template is delicate. For this reason, con- decontamination depends on the wavelength, energy,
tamination control is of utmost importance in designing and distance of the light source. The technologist must
a PCR procedure and laboratory setup. avoid skin or eye exposure to UV light, and UV light
Although genomic DNA (e.g., from hair, skin, or will also damage Plexiglas and some plastics, so labo-
ambient microorganisms) is a source of spurious PCR ratory equipment, including pipets, may be affected by
targets, the major cause of contamination is the presence extended exposure. Furthermore, the use of overhead
of PCR products from previous amplifications. Unlike UV room lights for decontamination is not very efficient
the relatively large and scarce genomic DNA, the small, for surface DNA decontamination due to the hazardous
highly concentrated PCR product DNA can aerosolize high intensity (4,000 microwatt-seconds/cm2) required
to damage DNA at the distances the light has to travel size determined by the primer placement. For instance,
from the ceiling to the bench surface. if two 20-base primers were designed to hybridize to
An effective method for decontamination and prepa- sequences flanking a target of 100 base pairs (bp), the
ration of the workspace is 10% bleach (7 mM sodium amplicon should be 140 bp in size. Any larger or smaller
hypochlorite). Frequently wiping bench tops, hoods, or amplicons would be due to mispriming, primer dimers,
any surface that comes in contact with specimen material or other artifacts of the reaction. For some procedures,
with dilute bleach or alcohol removes most DNA contam- these artifacts do not affect the interpretation of results
ination. As a practice in forensic work, before handling and can be ignored as long as they do not compro-
evidence or items that come in contact with evidence, mise the efficiency of the reaction. For other purposes,
gloves are wiped with bleach and allowed to air-dry. however, extraneous PCR products must be avoided or
An enzymatic method of contamination control is removed.
the dUTP–UNG system, which involves substitution of Misprimes are initially averted by good primer design
dUTP for dTTP in the PCR reagent master mix, result- and optimal amplification conditions. Even under the
ing in incorporation of dUTP instead of dTTP into the best conditions, however, misprimes can occur during
PCR product. Although some polymerase enzymes may preparation of the reaction mix. This is because Taq
be more or less efficient in incorporating the nucleo- polymerase has some activity at room temperature.
tide, the dUTP does not affect the PCR product in most While mixes are prepared and transported to the thermal
applications. At the beginning of each PCR, the enzyme cycler, the primers and template are in contact at 22°C
uracil-N-glycosylase (UNG) is added to the reaction to 25°C, a condition of very low stringency. Under these
mix. This enzyme will degrade any nucleic acid con- conditions, the primers can bind sequences other than
taining uracil, such as contaminating PCR product from their exact complements in the target, and the low-level
previous reactions. A short incubation period is added activity of Taq will extend them. These misprimed prod-
to the beginning of the PCR amplification program, ucts, then, are already present before the amplification
usually at 50°C for 2 to 15 minutes, to allow the UNG program begins. Hot-start PCR can be used to prevent
enzyme to function. The initial denaturation step in the this type of mispriming.
PCR cycle will degrade the UNG before synthesis of
the new products. Note that this system will not work
with some types of PCR, such as nested PCR (discussed
Advanced Concepts
later in the chapter), because a second round of ampli-
In addition to breaking the sugar-phosphate back-
fication requires the presence of the first-round product.
bone of DNA, UV light also stimulates covalent
The dUTP–UNG system is used routinely in quantita-
attachment of adjacent pyrimidines in the DNA
tive PCR procedures in which contamination control is
chain, forming pyrimidine dimers. These boxy
essential because the contaminant will affect the inter-
structures are the source of mutations in DNA
pretation of the amount of product.
in some diseases caused by sun exposure. DNA
Wipe tests are performed in some laboratories as a
repair systems remove these structures in vivo.
routine check or to confirm successful decontamination.
Loss of these repair systems is manifested in dis-
Filter paper is wiped on any exposed or touched sur-
eases such as xeroderma pigmentosum, Cockayne
faces in the pre-PCR setup, extraction, and amplification
syndrome, and trichothiodystrophy.14
areas. The paper is then placed in robust PCR buffer
with appropriate primers and subjected to amplification.
Any production of amplicon indicates the presence of Hot-start PCR can be performed in three different ways.
contamination. In one approach, the reaction mixes are prepared on ice
and placed in the thermal cycler after it has been pre-
warmed to the denaturation temperature. A second way
Mispriming
to perform hot-start PCR was used with older model
When PCR products are analyzed for size and purity by thermal cyclers that did not have heated lids and required
electrophoresis, the amplicon size should agree with the a vapor barrier over each reaction. A bead of wax was
placed in each reaction tube containing all components Gel containing DNA
of the reaction mix except enzyme and template. The
tubes were heated to 100°C to melt the wax and then
cooled to room temperature. The melted wax would float
to the top of the reaction mix and congeal into a phys-
ical barrier as it cooled. The remainder of the reaction Sieve
Supernatant
mix containing template and enzyme was then added on + alcohol
top of the wax barrier. When the tubes were placed in
Centrifuge
the thermal cycler, the wax melted at the denaturation
temperature, and the primers and template first came in DNA
contact at the proper annealing temperature. The floating precipitate
wax then served as an evaporation barrier as the reaction
proceeded. The third and most frequently used hot-start FIGURE 6.10 After gel electrophoresis, the gel band of PCR
product is excised with a clean scalpel or spatula. The gel is
method utilizes sequestered enzymes. These enzymes
disintegrated by centrifugation through a sieve, releasing the
are supplied in an inactive form, sequestered and inacti- DNA. The DNA in solution can then be separated from the gel
vated by monoclonal antibodies or by other proprietary fragments, precipitated with alcohol, and pelleted by a second
methods. The enzyme will not extend the primers until centrifugation.
it is activated by heat in the first denaturation step of the
PCR program, thereby preventing any primer extension
during the preparation of the reagent mix. with some post-PCR applications. Moreover, the buffers
Touchdown PCR is a modification of the PCR used for PCR may not be compatible with post-PCR
program used to enhance the amplification of the desired procedures. Amplicons free of PCR components are
PCR product. In this method, the PCR program begins conveniently prepared using spin columns (Fig. 6.11) or
with annealing temperatures higher than the optimal silica beads. The DNA binds to the column, and the rest
target primer-binding temperature. The annealing tem- of the reaction components are rinsed away by centrif-
perature is decreased by 1°C every cycle or every other ugation. The DNA can then be eluted from the column.
cycle until the optimal annealing temperature is reached. Even though columns or beads provide better recovery
Subsequent cycles are carried out at the optimal tem- than gel elution, they may not completely remove resid-
perature. Any difference in the annealing temperature ual primers or misprimed products.
will translate into an exponential advantage for correct Addition of alkaline phosphatase (AP) in combina-
versus mismatched primer binding.15 tion with exonuclease I (ExoI) is an enzymatic method
for removing nucleotides and primers from PCR prod-
ucts prior to sequencing or mutational analyses. During
PCR Product Cleanup
a 15- to 30-minute incubation at 37°C, AP dephosphor-
Sequence limitations in primer design or reaction condi- ylates unincorporated nucleotides, and ExoI degrades
tions may not completely prevent primer dimers or other the single-stranded primers. The enzymes must then
extraneous products. These unintended amplicons are be removed by extraction or inactivated by heating at
unacceptable for analytical procedures that demand pure 95°C for 5 minutes. This method is convenient because
product, such as sequencing or certain mutation analy- it is performed in the same tube as the PCR. It does not,
ses. A direct way to obtain a clean PCR product is to however, remove other buffer components.
resolve the amplification products by gel electrophore- In some post-PCR methods, such a small amount of
sis, cut out pieces of the gel containing desired bands, PCR product is added to the next reaction that resid-
and elute the PCR product. Agarose gel slices can be ual components of the amplification are of no conse-
digested with enzymes, such as β-agarase, or by incuba- quence, so no further cleanup of the PCR product is
tion with iodine, releasing the product DNA (Fig. 6.10). required. The choice of cleanup procedure or whether
Residual components of the reaction mix, such as cleanup is necessary at all will depend on the post-PCR
leftover primers and unused nucleotides, also interfere application.
Primer
PCR product
Salt
dNTP
Flip column
and
centrifuge
Centrifuge
FIGURE 6.11 PCR product cleanup in spin columns (left) removes residual components in the PCR mix. Amplicon DNA binds
to a silica matrix in the column while the buffer components flow through during centrifugation. The column is then inverted, and
the DNA is eluted by another centrifugation in low salt (Tris-EDTA) buffer.
and a second set can then detect the presence of a gene

PCR Modifications
that makes that organism resistant to a particular anti-
PCR has been adapted for various applications, several microbial agent. Multiplex PCR reagents and conditions
of which are used in the medical laboratory. Among the require more complex optimization. Target sequences
large and increasing number of PCR modifications are may not amplify with the same efficiency, and primers
the following methods that might be encountered in the may interfere with each other in binding to the target
clinical molecular laboratory. These methods are capable sequences. The conditions for the PCR must be adjusted
of detecting multiple targets in a single run (multiplex for the optimal amplification of all products in the reac-
PCR) using RNA templates (reverse transcriptase PCR) tion. Multiplexing primers is useful not only to detect
or such amplified products as templates (nested PCR) multiple targets but also to confirm accurate detection of
and quantifying starting template (quantitative PCR). a single target. Internal amplification controls are often
multiplexed with test reactions that are interpreted by
Multiplex PCR
the presence or absence of product. The control primers
More than one primer pair can be added to a PCR so
and targets must be chosen so that they do not inter-
that multiple amplifications are primed simultaneously,
fere or compete with the amplification of the test region.
resulting in the formation of multiple products. Multiplex
Internal amplification controls are the ideal for positive/
PCR is especially useful in typing or identification anal-
negative qualitative PCR tests.
yses. Individual organisms, from viruses to humans, can
be identified or typed by observing a set of several PCR Sequence-Specific PCR
products at once. Pathogen typing and forensic identifi- The strict requirement for complementarity of the
cation kits contain multiple sets of primers that amplify 3′ end of primers can be used to identify single-base
polymorphic DNA regions. The pattern of product sizes changes in the target DNA. By designing the forward
will be specific for a given type or individual. or reverse primer to end with a 3′ base complementary
Multiple organisms have been the target of multi- to the mutant sequence, the presence of the base change
plex PCR in clinical microbiology laboratories.16,17 One is detected by successful amplification. Although this
respiratory sample, for example, can be used to test for method is used frequently in the medical laboratory to
the presence of more than one respiratory virus.18 In a detect base changes in patient DNA, its high sensitivity
slightly different approach to testing for multiple targets, risks detection of mutations at very low levels that may
one set of primers can detect an infectious organism, not be clinically significant.19 Sequence-specific PCR is
one of the methods of human leukocyte antigen allele determined without long stretches of introns that might
analysis in tissue typing. complicate the analysis. Originally, RT PCR was per-
formed in two steps: cDNA synthesis and then PCR.
Reverse Transcriptase PCR Tth DNA polymerase, which has RT activity, and pro-
If the starting material for a procedure is RNA, the RNA prietary mixtures of RT and sequestered (hot-start)
may first be converted to double-stranded DNA, which DNA polymerase are components of one-step RT PCR
is a better template for amplification than single-stranded procedures.20 These methods are more convenient than
RNA. The conversion is accomplished through the the two-step procedure because RNA is added directly
action of reverse transcriptase (RT), an enzyme isolated to the PCR. The amplification program is modified to
from RNA viruses. This enzyme first copies the RNA include an initial incubation of 45°C to 50°C for 30 to
single strand into an RNA:DNA hybrid and then uses a 60 minutes, during which RT makes cDNA from RNA
hairpin formation on the end of the newly synthesized in the sample. The RT activity will then be inactivated,
DNA strand to prime synthesis of the complementary and the DNA polymerase activated, in the first denatur-
DNA strand, replacing the original RNA in the hybrid. ation step of the PCR procedure.
The resulting double-stranded DNA is copy or comple- Although RT PCR is a widely used and important
mentary DNA (cDNA). adjunct to molecular analysis, it is subject to the vulner-
Like other DNA polymerases, reverse transcriptase abilities of RNA degradation. As with other procedures
requires priming. Gene-targeted primers—oligo dT that target RNA, specimen handling is important for
primers or random hexamers—are most often used to accurate results.21 Methods have been described for the
prime the synthesis of the initial DNA strand. The yield RT PCR amplification of challenging specimens, such as
of cDNA will be relatively low using gene-targeted paraffin-embedded tissues; however, fixed specimens are
primers but highly specific for the target of interest. The difficult to analyze consistently.22
specific primers will prime cDNA synthesis only from
transcripts complementary to the primer sequences. Nested PCR
Oligo dT primers are 18-base-long single-stranded The increased sensitivity that PCR offers is very useful
polyT sequences that will prime cDNA synthesis only in clinical applications because clinical specimens are
from RNA with polyA tails (mRNA and some noncoding often limited in quantity and quality. Low levels of
RNA). The yield of cDNA will be higher with oligo dT target and the presence of interfering sequences can
primers than with target-specific primers because poten- prevent a regular PCR from working with the reliability
tially all polyA RNA could be included in the specimen. required for clinical applications. Nested PCR is a mod-
The highest yield of cDNA is achieved with random ification that increases the sensitivity and specificity of
hexamers or decamers. These are 6- or 10-base-long sin- the reaction.23-28
gle-stranded oligonucleotides with random sequences. In nested PCR, two pairs of primers are used to
The 6 or 10 bases will match and hybridize to random amplify a single target in two separate PCR runs. The
sites in the target RNA with some frequency, priming second pair of primers, designed to bind slightly inside
DNA synthesis. Random priming will generate cDNA of the binding sites of the first pair, will amplify the
from all RNA in the specimen. product of the first PCR in a second round of amplifica-
RT PCR is used to measure RNA expression profiles, tion. The second amplification will specifically increase
to detect rRNA, to analyze gene regions interrupted by the amount of the intended product. In semi-nested
long introns, and to detect microorganisms with RNA PCR, one of the second-round primers is the same as
genomes. For gene-expression analysis, the amount of the first-round primer (Fig. 6.12).
cDNA reflects the amount of transcript in the prepara- Several variations of nested and semi-nested PCR
tion. In other applications, genes that are interrupted by have been devised. For example, as shown in Figure
long introns can be made more available for consistent 6.12, the first-round primers can have 5′ sequences
amplification using cDNA versions lacking the inter- added (5′ tails) complementary to sequences used for
rupting sequences. cDNA is often used for sequenc- second-round primers. This tailed primer method is
ing because the sequence of the coding region can be valuable for multiplex procedures in which multiple
5′ 3′ 5′ 3′
3′ 5′ 3′ 5′
First-round product
Second-round product
FIGURE 6.12 Variations of nested PCR using nested primers (left), semi-nested second-round primers (right), and tailed first-
round primers. In nested PCR, a second reaction amplifies the product of the original reaction with primers binding sites present
on the amplicon. Both second-round products are binding within the amplicon. In semi-nested PCR, one primer binds within the
amplicon, and the other is identical to the first-round primer.
first-round primers may differ in their binding efficien- test material by preferential amplification over a known
cies. With the tailed primers, sequences complementary amount of competitor.25 These assays were also unreli-
to a single set of second-round primers are added to all able and inconsistent when test and internal control tem-
of the first-round products. In the second round, then, plates differed by more than 10-fold. They were most
all products will be amplified with the same primers. accurate with a 1:1 ratio of test and internal control,
Although this tailed primer procedure increases sensitiv- which required analysis of multiple dilutions of controls
ity in multiplex reactions, it does not increase specificity. for optimal results.
A very useful modification of the PCR process is
Real-Time (Quantitative) PCR real-time or quantitative PCR (qPCR).26,27 This method
Standard PCR procedures will indicate if a particular was first performed by adding ethidium bromide (EtBr)
target sequence is present in a clinical sample. For some to a standard PCR. Because EtBr intercalates into dou-
situations, though, the clinician is also interested in how ble-stranded DNA and fluoresces, it tracks the accumu-
much of the target sequence is present. Initially, there lation of PCR products during the PCR in real time, that
were several approaches to estimating the amount of is, as it is made. Detectable fluorescence in earlier cycles
starting template via PCR. The nature of amplification, of the amplification program indicates higher amounts
however, made calculating direct quantities of start- of starting template, whereas fluorescence appearing in
ing material complex. Strategies for quantifying start- later cycles indicates lower amounts of starting template.
ing material by quantifying the end products of PCRs This method more accurately reflects the amount of
utilized specialized internal controls, that is, known starting template. Furthermore, the quantitative measure-
quantities of starting material that were co-amplified ments are performed with the ease and rapidity of stan-
with the test template. These types of assays suffered, dard PCR without the addition of competitor templates
however, from primer incompatibilities and inconsistent or multiple internal controls. The rationale for qPCR
results. Another approach was to add competitor tem- is illustrated in Figure 6.13. Graphing the PCR cycles
plates at several known levels to assess the amount of (denaturation, annealing, extension) on the x-axis versus
107 copies
106 copies
105 copies
100
104 copies
103 copies
102 copies
101 copies
10
Rn
0.1
1 3 5 7 9 21 23 25 27 29 33 35 37 39 41 43 45 47 49
A Cycle
40.00
35.00
30.00
Threshold cycle (C)
25.00
20.00 Y = –3.345(x) + 38.808
15.00 R2 = 0.9983
10.00
5.00
0.00
1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07
B Starting quantity (copies/rxn)
FIGURE 6.13 A plot of the accumulation of PCR product over 50 cycles of PCR. (A) A sigmoid curve. The generation of fluo-
rescence occurs earlier with more starting template (solid lines) than with less (dotted lines). See Color Plate 1. The cycle number
at which fluorescence increases over a set amount, or fluorescence threshold, is inversely proportional to the amount of starting
material (B).
fluorescence generated (y-axis) yields an exponential (observe the ends of the amplification curves shown in
curve where the number of copies = 2N, with N being Fig. 6.13A). Using the fluorescent signal to detect the
the number of PCR cycles. The curve looks similar to a growing target copy number during the amplification
bacterial growth curve, with a lag phase, an exponential process, analysis in qPCR is performed in the expo-
(log) phase, a linear phase, and a stationary phase. nential phase of growth. Because the length of the lag
The exhaustion of reaction components and com- phase is inversely proportional to the amount of starting
petition between PCR product and primers during the template, fluorescence will reach exponential growth in
annealing step slow the PCR product accumulation after early cycles when a lot of target is present; when less
the exponential phase of growth until it finally plateaus. target is present, fluorescence will not reach exponential
In contrast to qPCR, analysis of PCR product by the growth until later cycles. With serial fold dilutions of
standard method occurs at the end of the PCR station- known positive standards, a relationship can be estab-
ary phase (endpoint analysis). In the endpoint analysis, lished between the starting target copy or cell number
products of widely different amounts of starting tem- and the cycle number at which fluorescence crosses a
plate are tested at the plateau where they are all the same threshold amount of fluorescence.
The use of nonspecific dyes to measure the accumula-

Advanced Concepts tion of product requires a clean PCR free of misprim-
ing and primer dimers because these artifactual products
The optimal threshold level is based on the back- will also generate fluorescence. More specific systems,
ground or baseline fluorescence and the peak flu- examples of which are described later in the chapter,
orescence in the reaction. Instrument software is have been devised that utilize probes designed to gener-
designed to set this level automatically. Alterna- ate fluorescence. The probes increase specificity by only
tively, the threshold may be determined and set yielding fluorescence when they hybridize to the target
manually. sequences.
The PCR cycle at which sample fluorescence crosses Advanced Concepts

the threshold is the threshold cycle, or CT. Plotting the
target copy number of the diluted standards against CT Fluorescence versus CT is an inverse relationship.
for each standard generates the graph shown in Figure The more starting material, the fewer cycles are
6.13B. Once this relationship is established, the start- necessary to reach the fluorescence threshold.
ing amount of an unknown specimen can be determined Samples that differ by a factor of 2 in the original
by the cycle number at which the unknown crosses the concentration of target are expected to be 1 cycle
fluorescence threshold. This method is applied to the apart, with the more dilute sample having a CT 1
quantification of DNA targets (qPCR) and RNA targets cycle higher than the more concentrated sample.
in reverse-transcriptase qPCR (RT-qPCR). The template Samples that differ by a factor of 10 (as in a
for RT-qPCR is cDNA. 10-fold dilution series) would be 3.3 cycles apart.
The first approach to qPCR utilized EtBr, which is The slope of a standard curve made with 10-fold
specific to double-stranded DNA. Dye is still used for dilutions, therefore, should be –3.3.
routine qPCR, except that EtBr has been replaced by
SYBR green, another dye specific to double-stranded
DNA. The advantage of SYBR green is its specificity TaqMan was developed from one of the first probe-
and robust fluorescence comparable to that of EtBr and based systems for quantifying cDNA by qPCR.28 This
its reduced toxicity. Both dyes bind and fluoresce specif- method exploits the natural 5′ to 3′ exonuclease activ-
ically in the double-stranded DNA product of the PCR ity of Taq polymerase to generate a signal. An early
(Fig. 6.14). version of the method reported by Holland et al. used
radioactively labeled probe and measured activity by the
release of radioactive cleavage fragments.29 The TaqMan
5′ 3′ procedure measures the fluorescent signal generated by
separation of fluorescent dye and quencher, a system
developed by Lee et al. that used a probe composed of
3′ 5′
a single-stranded DNA oligonucleotide complementary
to a specific sequence in the targeted region of the PCR
template.30 The probe is present in the reaction mix in
5′ 3′ addition to the specific primers that prime the DNA syn-
thesis reaction. The probe is chemically modified at its
3′ end so that it cannot be extended by the polymerase.
3′ 5′ The single-stranded DNA TaqMan probe is covalently
FIGURE 6.14 Non-sequence–specific dyes, such as EtBr and attached to a fluorescent dye on the 5′ end and another
SYBR green, bind to double-stranded DNA products of the dye or nonfluorescent molecule that pulls fluores-
PCR. As more copies of the target sequence accumulate, the cent energy from the 5′ dye (quencher) on the 3′ end31
fluorescence increases. (Fig. 6.15).
Primer R
R Probe Q
Q
5′
3′ 5′ 5′
3′ 5′
5′ 3′
5′ 3′
FIGURE 6.15 A TaqMan probe hybridizes to the target

sequences between the PCR primer-binding sites. The probe is
covalently attached to a fluorescent reporter dye (R) at the
5′ end and a quencher (Q) at the 3′ end.
R
Q
Advanced Concepts 5′
3′
3′
5′
EtBR is a planar molecule that intercalates 5′ 3′

3′ 5′
between the planar nucleotides in the DNA mol-
ecule. In doing so, it interferes with DNA metab- FIGURE 6.16 TaqMan signal fluorescence is generated when
olism and replication in vivo and is a mutagen. In Taq polymerase extends the primers and digests the probe and
contrast, SYBR green binds to the minor groove releases the reporter from the vicinity of the quencher.
of the double helix without disturbing the nucleo-
tide bases and thus does not upset DNA metabo-
lism to the extent that EtBr does.
([56]-carboxytetramethylrhodamine), or nonfluores-
cent quenchers, such as BHQ1, BHQ2 (Black Hole
Quenchers), and Eclipse. In the TaqMan system, the
As the polymerase proceeds to synthesize DNA on the quencher prevents fluorescence from the 5′ dye until
template to which the probe is hybridized, the natural they are separated during the synthesis reaction. As
exonuclease activity of Taq polymerase will degrade more copies of the template accumulate, more of
the probe into single and oligonucleotides, thereby the 5′ dye is accumulated throughout the reaction
removing the labeled nucleotide from the vicinity of the program.
quencher and allowing it to fluoresce (Fig. 6.16). Excess Another probe-based detection system, Molecu-
probe is present so that with every doubling of the target lar Beacons, measures the accumulation of product at
sequences, more probe binds and is digested, and more the annealing step in the PCR cycle.33 The signal from
fluorescence is generated. Probe design, like primer Molecular Beacons is detectable only when the probes
design, is important for a successful qPCR amplification, are bound to the template before displacement by the
as are the characteristics of the polymerase enzyme.32 polymerase. Here the probe is chemically modified so
The 5′ end of a TaqMan probe is labeled with one that it is not degraded during the extension step. Molec-
of a number of dyes with different “colors,” or peak ular Beacons are designed with a target-specific binding
emission wavelengths, of fluorescence, for example, sequence of approximately 25 bases flanked by a short,
FAM (6-carboxyfluorescein), TET (6-tetrachloroflu- approximately 5-base-long inverted repeat that will
orescein), HEX (6-hexachlorofluorescein), JOE (4′, form a stem and loop structure when the probe is not
5′-dichloro-2′, 7′-dimethoxy-fluorescein), Cy3, and bound to the template. There is a reporter fluorophore
Cy5 (indocarbocyanine). The probe is covalently (dye) at the 5′ end of the oligomer and a quencher at the
bound at the 3′ end with a quencher, such as DABCYL 3′ end. Until the specific product is present, the probe
(4-dimethylaminophenylazobenzoic acid) or TAMRA will form a hairpin structure that brings the fluorophore
Molecular Beacon
R Q R Q
R Q
3′ 5′
R Q
3′ 5′
R Q
FIGURE 6.17 A Molecular Beacon probe contains target-
specific sequences and a short, inverted repeat (~5 bp) that
hybridizes into a hairpin structure. The 5′ end of the probe has
a reporter dye (R), and the 3′ end has a quencher dye (Q).
in proximity with the quencher (Fig. 6.17). Fluorescence R Q

will occur on the binding of the probe to the denatured
template during the annealing step (Fig. 6.18). When the
primers are extended in the PCR, displacement of the
probe by Taq will restore the hairpin (nonfluorescent)
structure. Excess probe in the reaction mix will ensure
binding to the increasing amount of target. The amount
FIGURE 6.18 In the presence of target sequences, hybridiza-
of fluorescence, therefore, will be directly proportional tion of the probe will open the hairpin, moving the quencher
to the amount of template available for binding and from the reporter and allowing signal fluorescence, which
inversely proportional to the CT. doubles with every doubling of target sequences.
Scorpion-type primers are a variation of Molec-
ular Beacons.34,35 In contrast to free-labeled probes,
the PCR product will be covalently bound to the dye.
In this system, target-specific primers are tailed at the
5′ end with a sequence complementary to part of the and may be preferred for methods requiring fast cycling
internal primer sequence, a quencher, a stem-loop struc- conditions.36 Scorpions also produce a PCR product that
ture, and a 5′ fluorophore (Fig. 6.19). The fluorophore is covalently labeled with the fluorophore that can be
and the quencher are positioned so that they are jux- further analyzed by capillary electrophoresis.
taposed when the hairpin in the primer is intact. After Another frequently used system, fluorescent reso-
polymerization, the secondary structure of the primer is nance energy transfer (FRET), utilizes two specific
overcome by hybridization of the primer sequence with probes, one with a 3′ fluorophore (acceptor) and the
the target sequence, removing the fluorophore from the other with a 5′ catalyst for the fluorescence (donor),
quencher. This intramolecular system generates a signal that bind to adjacent targets.37 Examples of frequently
faster than the intermolecular Molecular Beacon strategy used donor–acceptor pairs are fluorescein–rhodamine,
D R
Primer
Primer
R Q
D R
R Q
Q
R D R
FIGURE 6.19 Scorpion primer/probes are primers tailed with

Molecular Beacon-type sequences. After extension of the
primer/probe, the target-specific sequences fold over to hybrid-
ize with the newly synthesized target sequences, separating the
reporter (R) from the quencher (Q). An advantage of this
system is the covalent attachment of fluorescent signal to the D R
PCR product, which is useful for further analysis, such as size
assessment by capillary electrophoresis.
fluorescein–(2 aminopurine), and fluorescein–Cy5. When FIGURE 6.20 FRET probes are separate oligomers, one
the donor and acceptor are brought within 1 to 10 nm covalently attached to a donor fluor (D) and one to an acceptor
(1 to 5 bases) through specific DNA binding, excitation or reporter fluor (R). The acceptor/reporter will fluoresce only
energy is transferred from the donor to the acceptor when both probes are bound next to one another on the target
sequences. As more target accumulates, more probes bind, and
(Fig. 6.20). The acceptor then loses the energy in the
more fluorescence is emitted.
form of heat or fluorescence emission. As with the
Molecular Beacons, the more template available for
binding of the probes, the more fluorescence will be
generated. Quantitative PCR lends itself to several
variations of technique, as exemplified previously. Advanced Concepts
These techniques can be further modified, for example,
using FRET probes with different sequences to distin- Internal controls are used with qPCR assays to
guish types of organisms or to detect mutations. FRET detect false-negative results in the event of ampli-
probes are also part of methods that use melt curves fication failure. Ideally, these controls should be
to detect gene mutations. As with standard PCR, RNA for RNA expression assays and plasmid for
many such methods have been devised for a variety DNA copy-number analyses.
of applications.
Promoter
Advanced Concepts Tailed primer
The ease of use and flexibility of qPCR has led RNA target
to the implementation of a variety of instruments, Reverse transcriptase
reagents, and analysis methods that have some-
times produced conflicting results. The Minimum
Information for Publication of Quantitative Real-
Time PCR Experiments (MIQE) guidelines recom- RNase II
mend the reporting of performance characteristics
ssDNA
for any qPCR assay used for publication.38,39 Per-
formance metrics include target specificity, PCR
efficiency, limit of detection, precision, dynamic
range, and either primer sequences or amplicon
context. These measurements can also be used to Primer
evaluate reagents. For clinical laboratories, they Reverse transcriptase
are included in the validation of qPCR methods to
cDNA
be used for medical laboratory testing.
Primer
FIGURE 6.21 The first step in transcription-based amplifica-

tion is the production of a complementary double-stranded
DNA copy of the RNA target. Synthesis is performed by
Transcription-Based Amplification Systems reverse transcriptase, which extends a primer that is tailed with
In a transcription-based amplification system (TAS), an RNA polymerase-binding site (promoter) sequence (purple).
RNA is the usual target instead of DNA. A DNA copy is The RNA:DNA hybrid is digested with RNase H, leaving the
synthesized from the target RNA, and then transcription single-stranded DNA, which is converted to a double strand
with a complementary primer. The DNA product will have a
of the DNA produces millions of copies of RNA. There
promoter sequence at one end.
are a number of commercial name variations of this pro-
cess: transcription-mediated amplification (TMA),
nucleic acid sequence–based amplification (NASBA),
and self-sustaining sequence replication (3SR). The original TAS procedure as just described had the
Kwoh and colleagues developed the first TAS in disadvantage that a heating step was required to denature
1989.40 TAS differs from other nucleic acid amplifi- the intermediate RNA:DNA hybrid product. The heat
cation procedures in that RNA is the target as well as also denatured the enzymes so that fresh enzyme had to
the primary product. In the original method of TAS, a be added after each denaturation step. The process was
primer carrying the binding site for RNA polymerase at simplified with the addition of RNase H derived from E.
its 5′ end and complementary to sequences in the target coli (Fig. 6.21). RNase H degrades the RNA from the
RNA is added to a sample of target RNA. The primer intermediate hybrid, eliminating the heating step. Thus,
anneals, and reverse transcriptase makes a DNA copy after synthesis of the DNA copy by reverse transcrip-
of the target RNA. Heat is used to denature the DNA– tase, the RNA strand is degraded by RNase H. Binding
RNA hybrid, and a second primer binds to the cDNA of the second primer and extension of the primer, pro-
and is extended by reverse transcriptase, producing dou- ducing double-stranded DNA by reverse transcriptase,
ble-stranded DNA. RNA polymerase derived from the is followed by transcription of the cDNA with T7 RNA
bacteriophage T7 then transcribes the cDNA, produc- polymerase (Fig. 6.22).
ing hundreds to thousands of copies of RNA. The tran- An additional modification and simplification of
scribed RNA can then serve as target RNA to which the the procedure came about with the discovery that the
primers bind and synthesize more cDNA. reverse transcriptase derived from avian myeloblastosis
RNA polymerase of organisms with DNA genomes, such as Mycobacte-

rium tuberculosis, is more sensitive than targeting the
cDNA
DNA because each bacterium, for example, has multiple
copies of RNA, whereas it has only one copy of DNA.
TMA, like NASBA, can also start with a DNA
cDNA target.41 For DNA, the sample is heated to denature the
RNA DNA, and the first primer anneals and is extended by
reverse transcriptase (which also has DNA-dependent
DNA polymerase activity in addition to having RNA-
dependent DNA polymerase activity). The RNA strand is
removed, and the second primer binds and is extended.
The DNA product has also incorporated the T7 RNA
polymerase binding site, which is on the 5′ end of the first
primer. Thus, T7 RNA polymerase transcribes the newly
replicated DNA into hundreds to thousands of RNA
copies. Detection of M. tuberculosis in smear-positive
respiratory samples, Chlamydia trachomatis in genital
Reverse transcriptase specimens, and HIV and cytomegalovirus (CMV) quan-
titation in blood are a few early applications of TAS.
Genomic Amplification Methods

In addition to amplification of single genes or transcripts,
methods have been devised to amplify all regions of
input DNA, or whole genomes. Whole-genome amplifi-
cation (WGA) methods are designed to survey all genes
or transcripts of an organism for the purpose of typing
of microorganisms or screening for particular genetic
lesions from limiting samples. Genomic amplification
FIGURE 6.22 The cDNA produced in the first step (Fig. differs from standard amplification methods in that the
6.21) serves as a template for RNA polymerase. Many copies primers used are not complementary to specific genetic
of RNA are synthesized, which are primed by a complemen- regions; rather, replication initiates at random sites
tary primer for synthesis of another RNA:DNA hybrid. After throughout the input nucleic acid (Fig. 6.23).42
RNase H degrades the RNA strand, the primer tailed with the Genomic amplification provides the ability to gener-
promoter sequences synthesizes another template, cycling back ate a lot of information from a very small amount of
into the system as a template for RNA polymerase.
virus (AMV) has inherent RNase H activity. Thus, TAS PCR

can be run with only two enzymes, AMV reverse tran-
scriptase and T7 RNA polymerase.
TAS has some advantages over PCR and other ampli-
fication procedures. First, in contrast to PCR, TAS is an WGA
isothermal process, negating the requirement for thermal
cycling and heat-stable enzymes to drive the reactions. FIGURE 6.23 In contrast to PCR, the object of whole-
Second, targeting RNA allows for the direct detection of genome amplification (WGA) is to copy all regions of a genomic
RNA viruses, for example, hepatitis C virus and human template. The WGA primers may be of random sequence or
immunodeficiency virus (HIV). Even targeting the RNA directed to highly repeated sequences in the genome.
starting material. In biological samples especially, the

target nucleic acid is not always clean, accessible, or in
optimal amounts for complex analyses, such as sequenc-
ing or microarray methods. The following methods are
approaches to genomic amplification applied to the ⫹ Adapters
typing of microorganisms and generation of multiple
genomic copies for genetic studies.
Whole-Genome Amplification
WGA can be performed using degenerative primers that
prime random synthesis throughout the target genome or
by nick translation. Degenerative primers are synthetic
single-stranded DNA sequences that vary with some fre- Emulsion
quency at each position where N = A, T, G, or C. In
Droplet
primer extension preamplification (PEP), this variation
occurs throughout the primer: N15–16. In degenerative FIGURE 6.24 Whole-genome DNA is fragmented, and the
oligonucleotide primer (DOP) amplification, the variable fragments are ligated to adapters that are complementary to one
sequences are flanked by a highly repeated sequence set of forward and reverse PCR primers. The adapted templates,
in the source DNA, for example, for human DNA: primers, and other PCR reagents in aqueous solution form an
emulsion with oil such that each aqueous droplet in the emulsion
CCGACTCGAGN6ATGTGG. In tagged-PCR (T-PCR),
contains a single template. Each of thousands of oil droplets then
primers have a 5′ sequence complementary to repetitive
serve as reaction chambers for unique products.
DNA followed by a random sequence of variable length:
CTCACTCTCANX.
Alternatively, whole-genome amplification can be
the other components of the PCR reaction (buffer, poly-
achieved by introducing single-strand breaks or nicks
merase enzyme, forward and reverse primers, dNTPs),
in the genomic DNA, with amplification primed by
are added to an oil surfactant mixture (Span 80, Tween
short random sequences (hexamers) or the 3′ ends of
80, Triton X-100, and mineral oil; Fig. 6.24). With stir-
the breaks.43 This is multiple-displacement amplifica-
ring, an emulsion is formed of 1 to 10 billion droplets,
tion because there is removal or denaturation of dou-
each of which can contain a single template in a tiny
ble-stranded DNA as extension proceeds. Amplification
volume of reaction buffer. When the emulsion is sub-
is performed using Phi29 DNA polymerase, a highly
jected to an amplification program, the droplets will act
processive enzyme—one that can displace and copy
as single reaction chambers producing many copies of
thousands of bases without losing contact with the
a specific sequence. After amplification, the emulsion is
template.
broken. The pooled products are used for a variety of
WGA has multiple applications, including compar-
genomic study applications, such as sequencing, array
ative genomic arrays, detection of single-nucleotide
studies, haplotyping, or detection of rare mutations.
polymorphisms, and analysis of minimal starting mate-
Solid-phase emulsion PCR was developed for next-
rial, such as DNA in plasma, single-cell analysis, and
generation sequencing and other high-throughput tech-
ancient DNA samples.
nologies that have driven the development of equally
high-throughput PCR technologies. In this method,
Emulsion PCR
templates ligated to a universal primer sequence are
Emulsion PCR is designed to simultaneously amplify mixed with bead-immobilized primers and other reac-
thousands of specific templates in a single reaction, tion components in the water-in-oil emulsion (Fig. 6.25).
producing a set of specific products, or library.44 In Aqueous droplets in the oil ideally would capture a single
this method, the ends of fragmented template DNA are template and a single primer-coupled bead. During the
ligated to universal primer sequences and, along with PCR reaction, the primers are extended, producing a
primers will anneal under nondenaturing buffer condi-

tions. After extension of the primer, denaturing condi-
tions are introduced by the addition of formamide to
the reaction mixture. The template and reaction com-
ponents are then removed by washing, and annealing
conditions are reestablished chemically, allowing the
Emulsion attached copy of the template to anneal to the immo-
bilized reverse primer. The reverse primer is extended,
forming a bridge between the two immobilized primers.
Droplet Bead
The process is repeated for 35 cycles, producing approx-
imately 1,000 copies of the template sequence in a tiny
region of the surface. The double-stranded bridges are
then denatured, resulting in a localized clone or polony
of single-stranded complementary molecules. To avoid
re-annealing, a cleavable site located on one primer
(either forward or reverse) allows removal of one of
the complements (Fig. 6.27). For DNA sequence appli-
cations, the remaining strand is chemically blocked at
the free 3′ end using terminal DNA transferase and a
dideoxynucleotide molecule that cannot be extended.
Sequencing primer is then annealed to the template for
sequencing.
FIGURE 6.25 For solid-phase ePCR, the template is pre-
pared as described in Figure 6.25. Many copies of one primer
are covalently attached to a microsphere (bead). In the emul- Arbitrarily Primed PCR
sion droplets, a single template will be copied into immobi- In arbitrarily primed PCR, also known as randomly
lized products on the beads. After the PCR reaction, the amplified polymorphic DNA or random amplification
emulsion is broken, the beads are released, and the comple-
of polymorphic DNA (RAPD), short primers (10 to 15
mentary strands are washed away, leaving many copies of
bases) with random sequences are used to amplify arbi-
immobilized single strands on each bead.
trary regions in genomic DNA under low-stringency
conditions.45,46 With this method, PCR products are gen-
erated without knowing the sequence of the target or
double-stranded copy from the template attached to the
targeting a specific gene. In contrast to standard PCR
bead. When the emulsion is broken, the PCR products
where only one or a few known products are gener-
are denatured, washing away the complementary strand
ated, multiple products are generated depending on how
and leaving the single-stranded sequencing templates
many times a short sequence appears in the genome
attached to the beads. These templates can then be sub-
(Fig. 6.27). Arbitrarily primed PCR has been used pri-
jected to massive parallel (next-generation) sequencing
marily in the epidemiological typing of microorgan-
technologies.
isms. Similar band patterns obtained from performing
PCR with the same arbitrary primers indicate that two
Surface Amplification (Bridge PCR)
organisms are the same or similar. A disadvantage of this
Surface amplification is an isothermal, genomic PCR method, however, is that reproducibility between runs
method used in high-throughput sequencing technol- is not very good, such that two organisms that had the
ogies. In this method, forward and reverse primers are same PCR product pattern on one day could have two
immobilized on a solid support in a flow cell (Fig. 6.26). different patterns and look like two different organisms
Fragmented or preamplified template is denatured, and when amplified on another day. Other methods such as
those single strands complementary to the immobilized mass spectrometry and rRNA sequencing provide more
Reverse
primer Hybridize
Forward template Extension Denature
primer
with
restriction
site
Anneal Extension
Cut with
Cycle Denature endonuclease
Anneal
sequencing
Block primer
3' ends
and
anneal
sequencing
primer
FIGURE 6.26 Bridge PCR produces immobilized single-stranded products from immobilized forward and reverse primers. One
primer is designed to contain a single-strand endonuclease recognition site. After amplification, the PCR products form bridges
from the forward to the reverse primers so that upon denaturation, the single strands are also immobilized. Endonuclease digestion
will remove one complement of single strands so that the remaining population of immobilized single strands is identical. The
strands are then subjected to further analysis, such as sequencing.
reproducible typing results; however, RAPD offers a rel- Ligase Chain Reaction
atively inexpensive alternative.
LCR was a method for amplifying synthetic primers/
probes complementary to target nucleic acid. Similar to
PROBE AMPLIFICATION PCR, the entire target sequence had to be known in order
to prepare the oligonucleotide primers for LCR. In PCR,
In probe amplification procedures, the number of target there is a distance between the primers of hundreds to
nucleic acid sequences in a sample is not changed. thousands of bases that is part of the amplified sequence.
Rather, synthetic probes that are specific to the target In LCR, by contrast, the primers are bound immediately
sequences bind to the target where the probes them- adjacent to each other. Instead of DNA polymerase syn-
selves are amplified. There are three major procedures thesizing complementary DNA by extending the primers
that involve the amplification of probe sequences: ligase as occurs in PCR, DNA ligase was used in LCR to ligate
chain reaction (LCR), strand displacement amplification the adjacent primers together. The ligated primers then
(SDA), and Qβ replicase. served as a template for the annealing and ligation of
M 1 2 3 4 M …GTACTCTAGCT… …GTACTCTAGCT…
A
T
C A
…CATGAGATCGA… …CATGAGATCGA…
Ligase Ligase
A
C A
T
A
C
A
A
C
T
A
C
FIGURE 6.27 RAPD or arbitrarily primed PCR yields many

products per PCR reaction. The band pattern produced by these
FIGURE 6.28 Ligase chain reaction generates a signal by
repeated ligation of probes complementary to specific
fragments reflects the nature of the DNA template. Lanes 1 and
sequences in the test DNA. One complementary oligomer is
3 are products from very similar templates, which are different
covalently attached to biotin for immobilization (square), and
from those in lanes 2 and 4. M, molecular-weight markers.
one has a signal-producing molecule (circle). The two oligo-
mers will be ligated together only if the sequence of the target
additional primers. Because the product of LCR was is complementary (left). The oligomers captured on a solid
ligated primer, LCR was a method of probe amplifica- substrate by streptavidin will generate a signal. If the sequence
of the target is not complementary (right), the captured probe
tion rather than target amplification because the copy
will not yield a signal.
number of target molecules did not change.
LCR required a thermal cycler to change the tem-
perature to drive the different reactions. In LCR the In SDA the major amplification products are the probes.
reaction was heated to denature the template. When There are two stages to the SDA process. In the first
the temperature was cooled, the primers annealed if the stage (target generation), the target DNA is denatured
complementary sequence was present, and a thermo- by heating to 95°C. At each end of the target sequence,
stable ligase joined the two primers (Fig. 6.28). Even two primers bind close to each other, an outer and
a 1-bp mismatch at the ligation point prevented ligation an inner primer (Fig. 6.29). The inner primers have a
of the primers. LCR was used to detect point mutations 5′ tail containing a recognition sequence for the restric-
in a target sequence. The DNA mutation that occurs in tion endonuclease enzyme, HincII. Exonuclease-deficient
the beta globulin of patients with sickle cell disease, as DNA polymerase derived from E. coli DNA polymerase
compared with normal beta globulin, was one of the first I extends all of the primers, incorporating a modified
applications of LCR.47 LCR in the clinical laboratory nucleotide, 2′-deoxyadenosine 5′-O-(1-thiotriphosphate)
has mostly been replaced by other methods; however, (dATPaS), into the nucleotide mix. As the outer primers
new applications of the technology have been proposed are extended, they displace the products formed by the
using FRET for detection of DNA single-nucleotide extension of the inner primers. A second set of outer and
changes,48 quantification of RNA,49 and DNA methyla- inner primers then binds to the displaced inner primer
tion analysis.50 products, and DNA polymerase extends the complemen-
tary primers, producing four double-stranded products
(probes). These probes are the target DNA for the next
Strand Displacement Amplification
stage of the process.
Strand displacement amplification (SDA) is an isother- The second stage of the reaction is the exponential
mal amplification process; that is, after an initial dena- probe amplification phase using HincII (Fig. 6.30). When
turation step, the reaction proceeds at one temperature.51 the restriction enzyme is added to the double-stranded
Probe with
restriction site
Primer
Modified
nucleotides
Displaced
strand
FIGURE 6.29 The first stage of SDA is the denatur-
ation of the double-stranded target and annealing of
primers and probes tailed with sequences including a
restriction enzyme site (only one strand of the initial
Displaced target is shown). A second reaction copies the probe,
strand targets incorporating dATPαS and thereby inactivating the
Inactive restriction site restriction site on the copied strand. This species is
(due to incorporation the target for amplification in the second stage of the
of modified nucleotides) reaction.
probe DNA, only one strand of the probe will be cut Qβ Replicase
due to the dATPαS introduced in the extension reaction.
Qβ replicase is another method for amplifying probes
This forms a nick in the DNA that is extended by DNA
that have specificity for a target sequence. The method
polymerase, simultaneously displacing the opposite
is named for the major enzyme that is used to amplify
strands. The nicking and extension form single-stranded
probe sequences. Qβ replicase is an RNA-dependent
probes and regenerate the restriction site. The nicking/
RNA polymerase from the bacteriophage Qβ.53 The
extension reaction can repeat on the initial probe as well
target nucleic acid in this assay can be either DNA
as the probes generated in the reaction. With the enzyme
(which must first be denatured) or RNA.
nicking reaction, strand displacement does not require
The target nucleic acid is added to a reaction mix
denaturation of the double-stranded probes. Thus, the
containing reporter probes, which are RNA molecules
iterative process takes place at about 52°C without tem-
(midivariant RNA) that have specificity for the target
perature cycling. Addition of a fluorogenic probe to the
sequence and also contain a promoter sequence that is
reaction produces a fluorescent signal that corresponds
recognized by the Qβ replicase. The reporter probes are
to the amount of amplified target.
allowed to hybridize to the template. The template with
The SDA process was first widely applied to detec-
bound probes is immobilized using polyG-tailed capture
tion of M. tuberculosis.52 Methods have been designed
probes (Fig. 6.31, capture probe A) and magnetic beads
to test for M. tuberculosis, C. trachomatis, and Neisseria
covalently attached to polyC sequences. With a magnet
gonorrhoeae.
applied to the outside of the wells, the unbound reporter
molecules are washed away. The template–probe com-
Advanced Concepts plexes are released from the polyC magnetic bead by
denaturation and hybridized to a polyA capture probe
For SDA of RNA or other single-stranded targets, (capture probe B). The complex is then hybridized to
the initial heat denaturation step is not necessary. a polyT paramagnetic bead. After a series of washes
The inner primer tailed with the restriction site to remove unbound reporter probe, the template–probe
is annealed to the 3′ end of the target, forming a complex is again released from the magnetic bead.
double-stranded probe that will enter the iterative For the amplification step, the midivariant RNA
restriction/strand displacement reactions. probe-bound template is mixed with the Qβ replicase,
which replicates the midivariant RNA molecules. This
Nick Capture probe A Reporter probe

G
G
G
G
First
G hybridization
Magnetic
bead Target RNA
G
C G
Magnet C G Capture, wash
C G
C G
C
G
C G
C G
C
C
G Release
G
C
Nick
Capture probe B
A Second
A
A
A
hybridization
A
Nick A
T A
T A
T A
T A
T
Reversible
target capture
Nick and washes
T
T A
FIGURE 6.30 In the second phase of SDA, the target T
T
T
A
A
A
sequence is nicked by the restriction enzyme, generating a sub- A
strate for the polymerase, which extends the nick, displacing

the opposite strand, and regenerates a new template for nicking. Q␤ replicase
Copies of the displaced strands collect as the reaction pro-
ceeds. The reaction cycles by the strands are nicking and Amplification
extension, without requirement for heat denaturation of the
double-stranded DNA template.
replication is very efficient; it generates 106 to 109 RNA FIGURE 6.31 The Qβ replicase method proceeds through a
series of binding and washing steps. Probe bound to the puri-
molecules (probes) in less than 15 minutes. Because so
fied template is then amplified by Qβ replicase. The resulting
many RNA molecules are produced, product detection
RNA can be detected by fluorometry using propidium iodide
can be achieved by colorimetric as well as real-time flu- as a fluorescent label of the synthesized probe or by chromo-
orogenic methods. Qβ replicase has been replaced by genic methods.
other methods for most medical lab applications. It was
used primarily to amplify the nucleic acid associated
with infectious organisms, particularly mycobacteria,
Chlamydia, HIV, and CMV.
SIGNAL AMPLIFICATION sequences in the target molecules as well as to sequences

in amplifier probes.
In signal amplification procedures, there is no change in In the first-generation assay, the extender probes bind
the number of target or probe sequences; instead, large to a tree-like bDNA amplifier probe, which in turn binds
amounts of signal are bound to the target sequences that multiple alkaline phosphatase-labeled nucleotides. Eight
are present in the sample. Signal amplification proce- multimers or amplifiers, each with 15 branches, bind to
dures are inherently better at quantifying the amount of each extender probe bound to the target. In the second-
target sequences present in the clinical sample. Several and third-generation assays, the extender probes bind pre-
signal amplification methods are available commercially. amplifiers, which in turn bind 14 to 15 amplifiers, each
with the capacity to bind multiple alkaline phosphatase–
labeled oligonucleotides (Fig. 6.33). Dioxetane is added
Branched DNA Amplification
as the substrate for the alkaline phosphatase, and chemi-
In branched DNA (bDNA) amplification,54 a series of luminescence is measured in a luminometer. This system
short oligomer probes is used to capture a single target has a detection limit of about 50 target mol/mL.55
nucleic acid molecule. Additional extender probes bind There are several advantages to the bDNA method.
to the target nucleic acid and then to multiple reporter First, in the bDNA assay there is less risk of carry-
molecules, loading the target nucleic acid with signal. over contamination resulting in a positive test than in
For the bDNA signal amplification procedure, target PCR.56 Second, multiple capture and extender probes
nucleic acid released from the cells is denatured (if DNA can be incorporated that detect slightly different target
is the target; this method also works with RNA). The
target nucleic acid binds to capture probes that are fixed
to the plate well (Fig. 6.32). Extender or preamplifier Amplifiers
probes then bind to the captured target. The extender
probes have sequences that are complementary to
Amplifiers
Extender probes
Preamplifier
Target RNA or DNA
Capture
Extender probes
probes
Solid support
Target RNA or DNA
Capture
probes
FIGURE 6.32 Branched DNA signal amplification of a
single target. The target is captured or immobilized to a solid
Solid support
support by capture probes, after which extender probes and
blocking probes create a stable cruciform structure with the
amplifiers. Each amplifier has hybridization sites for 8 to 14 FIGURE 6.33 Second-generation bDNA assays use extender
branches, which in turn bind substrate molecules for alkaline probes that bind multiple amplifiers, increasing the signal
phosphatase. intensity and improving limits of detection.
sequences, as occurs with different isolates of hepatitis chemiluminescence is measured. The hybrid capture
C virus and HIV. By incorporating different probes that assay is considered a signal amplification assay because
recognize slightly different sequences, multiple gen- the amount of target DNA is not amplified; rather, the
otypes of the same virus can be detected by the same DNA is isolated bound to RNA and is recognized by
basic system. Finally, the requirement for probes to multiple antibodies to the target/probe hybrid molecule.
bind multiple sequences in the same target increases the
specificity of the system. It is highly unlikely that all
Cleavage-Based Amplification
of the required probes would bind nonspecifically to an
unrelated target and produce a signal. The bDNA signal Cleavage-based amplification detects target nucleic
amplification assay has been applied to the qualitative acids by using a series of overlapping probes that bind to
and quantitative detection of hepatitis B virus, hepatitis the target DNA. Cleavase is a bacterial enzyme that rec-
C virus, and HIV-1. By replacing the plate support with ognizes overlapping sequences of DNA and makes a cut
beads, the assay has been combined with the bead array (cleaves) in the overlapping region. In vivo, this activity
technology to provide a multiplex system that can detect is most likely important in repairing DNA. Applications
100 different targets in a single sample.57 for this form of amplification include DNA polymor-
phisms, such as factor V Leiden mutation detection,60,61
and infectious diseases, such as hepatitis C virus (HCV)
Hybrid Capture Assays
and HPV genotyping.62,63
The hybrid capture assays were marketed primarily for To start the amplification, the target nucleic acid is
the detection and molecular characterization of human mixed with invader and signal probes (Fig. 6.35). The
papillomavirus (HPV) in genitourinary specimens.58,59 invader probe and the signal probes bind at the target,
They are also used for the detection of hepatitis B virus with the 5′ end of the signal probe overlapping with
and CMV. For these assays, target DNA released from the invader probe. Cleavase recognizes this triple-he-
cells is bound to single-stranded RNA probes (Fig. 6.34). lix overlap and cleaves the signal probe, which can act
The DNA:RNA hybrid has a unique structure that is rec- as an invader probe in the next step of the reaction.
ognized by antibodies. Antibodies bound to the surface In the second step, a FRET probe is added that has
of a microtiter well then capture the DNA:RNA hybrids. sequences complementary to the cleaved signal probe.
Double-stranded DNA or single-stranded RNA will not The 5′ end of the FRET probe has a reporter molecule
bind to these antibodies. Captured hybrids are detected that is located in proximity to a quencher molecule. As
by the binding of alkaline phosphatase–conjugated a result, the intact FRET probe does not produce signal.
secondary antibodies to the DNA:RNA hybrid anti- The signal probe (now an invader probe) binds to the
bodies in a typical sandwich assay. A light-producing FRET probe, producing an overlapping region that is
substrate for the alkaline phosphatase is added, and recognized by Cleavase. When Cleavase cuts the FRET
Capture
antibodies
FIGURE 6.34 Hybrid capture starts

with hybridization of the RNA probe
to the denatured DNA target. The
RNA:DNA hybrid is then bound by
hybrid-specific immobilized antibodies.
A secondary antibody bound to alkaline
phosphatase generates signal in the
presence of a chemiluminescent sub- Denatured RNA Hybrid Antibody Substrate
strate (right). DNA target probe conjugate
Test probe Test probe

Flap Flap
A A
T C FIGURE 6.35 Invader assays exploit the substrate

Mutation present Cleavage Normal sample (no cleavage) requirements of the enzyme. A cleavable substrate is
A formed by the hybridization of two probes and tem-
Complex formation plate (top left). This structure will not form if the
F Q
probe does not match the template (top right). If the
proper substrate is formed, the enzyme removes part
of one probe, and that fragment forms another cleav-
A
able complex with the fluorescently labeled reporter
probe (bottom left). Repeated binding and cleavage
Cleavage Fluorescence in plate well indicates amplify the signal. In a plate format, only those
F
presence of test sequence wells with a template complementary to the test
in the template probe will produce fluorescent signal.
probe in the overlapping region, it releases the reporter DNA-RNA-DNA probe

molecule from the quencher, resulting in the production
of signal. The amount of signal can be quantified and R
related directly to the amount of target molecules in Q
the sample. These reactions are carried out in a 96-well
plate format, and the signal is detected on a plate reader. DNA target
R Q
Cycling Probe
RNase
In the cycling probe method of amplification, target
sequences are detected using a synthetic probe consist- R Q
ing of sequences of DNA and RNA arranged in a DNA–
RNA–DNA sandwich sequence carrying a reporter dye
at one end and a quencher dye at the other. After the R
probe binds to the target nucleic acid (Fig. 6.36), RNase Q
H cleaves the RNA from the middle of the probe. The
loss of the RNA sequences lowers the hybridization
FIGURE 6.36 Cycling probe produces fluorescence only
temperature of the probe, and the DNA portions of the when the RNA probe binds to the DNA template. The
probes leave the template. When the probe is released, RNA:DNA hybrid formed by the probe bound to the template
the reporter and quencher dye are separated, allowing is a substrate for RNase H, which digests the RNA probe
fluorescence to escape from the reporter. The template and releases the reporter dye (R) from the vicinity of the
remains available for additional probe hybridization. quencher (Q).
Because the cyclic denaturation of the probe depends on
the digestion of the RNA portion, the method is isother-
mal. The amount of fluorescence from the reporter dye
(produced when the target is present) is measured as an with antimicrobial resistance in bacteria, such as meth-
indication of the presence of target molecules. Alterna- icillin resistance (mecA) in Staphylococcus aureus and
tively, the presence of chimeric probes that remain when vancomycin resistance (vanA and vanB) in Enterococ-
target sequences are not present can also be measured. cus,64-66 and in the detection of other organisms, such as
This method has been used to detect genes associated herpesvirus and Histoplasma capsulatum.67,68
c. 302
STUDY QUESTIONS d. 30/2
1. A master mix of all components (except template) 8. What are the three steps of a standard PCR cycle?
necessary for PCR contains what basic ingredients?
9. Which of the following is a method for purifying a
2. The final concentration of Taq polymerase is to be PCR product?
0.01 units/μL in a 50-μL PCR. If the enzyme is a. Treating with uracil N glycosylase
supplied at 5 units/μL, how much enzyme would b. Adding divalent cations
you add to the reaction? c. Putting the reaction mix through a spin column
a. 1 μL d. Adding DEPC
b. 1 μL of a 1:10 dilution of Taq
c. 5 μL of a 1:10 dilution of Taq 10. In contrast to standard PCR, real-time PCR is
d. 2 μL a. quantitative.
b. qualitative.
3. Primer dimers result from c. labor-intensive.
a. high primer concentrations. d. sensitive.
b. low primer concentrations.
c. high GC content in the primer sequences. 11. In real-time PCR, fluorescence is not generated by
d. 3′ complementarity in the primer sequences. which of the following?
a. FRET probes
4. Which control is run to detect contamination? b. TaqMan probes
a. Negative control c. SYBR green
b. Positive control d. Tth polymerase
c. Molecular-weight marker
d. Reagent blank 12. Prepare a table that compares PCR, LCR, bDNA,
TMA, Qβ replicase, and hybrid capture with regard
5. Nonspecific extra PCR products can result from to the type of amplification, target nucleic acid,
type of amplicon, and major enzyme(s) for each.
a. mispriming.
b. high annealing temperatures. 13. Examine the following sequence. (The
c. high agarose gel concentrations. complementary strand is not shown.) You
d. omission of MgCl2 from the PCR. are devising a test to detect a mutation at the
underlined position.
6. Using which of the following is an appropriate way
to avoid PCR contamination? 5′TATTTAGTTA TGGCCTATAC ACTATTTGTG
AGCAAAGGTG ATCGTTTTCT GTTTGAGATT
a. High-fidelity polymerase
TTTATCTCTT GATTCTTCAA AAGCATTCTG
b. Hot-start PCR
AGAAGGTGAG ATAAGCCCTG AGTCTCAGCT
c. A separate area for PCR setup
ACCTAAGAAA AACCTGGATG TCACTGGCCA
d. Fewer PCR cycles
CTGAGGAGC TTTGTTTCAAC CAAGTCATGT
GCATTTCCAC GTCAACAGAA TTGTTTATTG
7. How many copies of a target are made after 30
TGACAGTTAT ATCTGTTGTC CCTTTGACCT
cycles of PCR?
TGTTTCTTGA AGGTTTCCTC GTCCCTGGGC
a. 2 × 30 AATTCCGCAT TTAATTCATG GTATTCAGGA
b. 230 TTACATGCAT GTTTGGTTA AACCCATGAGA
TTCATTCAGT TAAAAATCCA device with integrated microwave heating and air impingement
GATGGCGAAT3′ cooling. Lab Chip 2010;10:1725–1728.
9. Cone R, Fairfax MR. Protocol for ultraviolet irradiation of sur-
Design one set of primers (forward and reverse) faces to reduce PCR contamination. Genome Research 1993;3:
to generate an amplicon containing the underlined s15–s17.
10. Jelden K, Gibbs SG, Smith PW, Hewlett AL, Iwen PC, Schmid
base. KK, Lowe JJ. Ultraviolet (UV)-reflective paint with ultraviolet
The primers should be 20 bases long. germicidal irradiation (UVGI) improves decontamination of nos-
ocomial bacteria on hospital room surfaces. Journal of Occupa-
The amplicon must be 100 to 150 bp in size. tional and Environmental Hygiene 2017;14(6):456–460.
11. Klaschik S, Lehmann LE, Raadts A, Hoeft A, Stuber F. Compar-
The primers must have similar melting ison of different decontamination methods for reagents to detect
temperatures (Tm), +/− 2°C. low concentrations of bacterial 16S DNA by real-time-PCR.
Molecular Biology 2002;22:231–242.
The primers should have no homology in the last 12. Meier A, Persing DH, Finken M, Bottger EC. Elimination of
three 3′ bases. contaminating DNA within polymerase chain reaction reagents:
implications for a general approach to detection of uncultured
a. Write the primer sequences 5′→3′ as you pathogens. Journal of Clinical Microbiology 1993;31:646–652.
would if you were to order them from the DNA 13. Fox J, Ait-Khaled M, Webster A, Emery VC. Eliminating PCR
synthesis facility. contamination: is UV irradiation the answer? Journal of Virologi-
cal Methods 1991;33:375–382.
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designed. genesis. Frontiers in Bioscience 2002;7:1024–1043.
15. Korbie D, Mattick JS. Touchdown PCR for increased speci-
14. How does nested PCR differ from multiplex PCR? ficity and sensitivity in PCR amplification. Nature Protocols
2008;3:1452–1456.
16. Tzanakaki G, Tsopanomichalou M, Kesanopoulos K, Matzourani
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Chapter 7
Chromosomal Structure
and Chromosomal Mutations
Outline Objectives
CHROMOSOMAL STRUCTURE AND ANALYSIS 7.1 Define mutations and polymorphisms.
Chromosomal Compaction and Histones 7.2 Distinguish the three types of DNA mutations:
Chromosome Morphology genome, chromosomal, and gene.
Visualizing Chromosomes 7.3 Describe chromosomal compaction and the
DETECTION OF GENOME AND CHROMOSOMAL MUTATIONS proteins involved in chromatin structure.
Karyotyping 7.4 Diagram a human chromosome, and locate the
Fluorescence In Situ Hybridization centromere, telomere, q arm, p arm, and express
Interphase FISH ideogram locations.
Metaphase FISH 7.5 Illustrate the different types of structural mutations
COMPARATIVE GENOME HYBRIDIZATION that occur in chromosomes.
7.6 Describe how karyotypes are made and state
the karyotype designation of a normal male and
female.
7.7 Identify the chromosomal abnormality in a given
karyotype.
7.8 Interpret results from interphase and metaphase
FISH analyses.
7.9 Distinguish between the effects of balanced and
unbalanced translocations on an individual and
the individual’s offspring.
7.10 Interpret the results of comparative genome
hybridization showing an amplification or
deletion.
179
The human genome is all of the genes found in a single

individual. The human genome consists of 2.9 billion TABLE 7.1 Approximate Sizes of Human
nucleotide base pairs of DNA organized into 23 chro- Chromosomes in Base Pairs
mosomes. As diploid organisms, humans inherit a
haploid set of genes (23 chromosomes) from each Chromosome Millions of Base Pairs
parent, so humans have two copies of every gene 1 249
(except for some on the X and Y chromosomes). Each
chromosome is a double helix of DNA, ranging from 2 242
246 million nucleotide base pairs in length in chro- 3 198
mosome 1 (the largest) to 48 million nucleotide base
pairs in chromosome 21 (Table 7.1). Genetic informa- 4 191
tion is carried on the chromosomes in the form of the
5 181
order or sequence of nucleotides in the DNA helix. A
phenotype is a trait or group of traits resulting from 6 171
transcription and translation of these genes. The gen-
7 159
otype is the DNA nucleotide sequence responsible for
a phenotype. 8 146
Genotypic analysis is performed to confirm or predict
9 141
phenotype. In the laboratory, some changes in chromo-
some structure and chromosome number can be observed 10 135
microscopically. Mutations at the nucleotide-sequence
11 135
level are detected using biochemical or molecular
methods. Alterations of the DNA sequence may affect 12 133
not only the phenotype of an individual but the progeny
13 115
of that individual as well. The latter, heritable changes
are the basis for prediction of the phenotype in the next 14 107
generation. The probability of inheritance of a pheno-
15 102
typic trait can be estimated using the logical methods of
Mendel’s laws of genetics and statistics. 16 90
A transmissible (inheritable) change in the DNA
sequence is a mutation or polymorphism. Although these 17 83
terms are sometimes used interchangeably, they do have 18 78
slightly different meanings based on population genet-
ics. A DNA sequence change that is present in a rela- 19 59
tively small proportion of a population is a mutation. 20 63
The more general term variant may be used, particularly
to describe inherited or somatic sequence alterations, 21 48
reserving the term mutation for rarer, usually somatic 22 49
changes, for example, changes found only in tumor
tissue. A variant that is present in at least 1% to 2% of X 155
a population is considered a polymorphism. Both muta- Y 59
tions and polymorphisms may or may not produce phe-
notypic differences.
Polymorphisms are casually considered mutations
that do not severely affect phenotype; this is gener-
ally true because any negative effect on survival and
reproduction limits the persistence of a genotype in a
Chapter 7 • Chromosomal Structure and Chromosomal Mutations 181
population. Some polymorphisms, however, are main- CHROMOSOMAL STRUCTURE AND ANALYSIS
tained in a population through a balance of positive
and negative phenotype. The classic example is sickle Chromosomal Compaction and Histones
cell anemia, a condition caused by a single-base sub-
stitution in the gene that codes for hemoglobin. The An important concept in the understanding of chromo-
alteration is regarded as a mutation, but it is really a somes is that chromosome behavior is dependent on
balanced polymorphism. In addition to causing abnor- chromosome structure as well as DNA sequence.1 Genes
mal red blood cells, the genetic alteration results in with identical DNA sequences will behave differently,
resistance to infection by Plasmodium species, that depending on the chromosomal location or the surround-
is, resistance to malaria. The beneficial trait provides ing nucleotide sequence of the gene. It is a well-known
a survival and reproductive advantage that maintains phenomenon that a gene inserted or moved into a differ-
the polymorphism in a relatively large proportion of ent chromosomal location may be expressed (transcribed
affected populations. Examples of benign polymor- and translated) differently than it was in its original
phisms with no selective advantage are the ABO blood position. This is called the position effect. Further-
groups and the major histocompatibility complex and more, different sequences can have the same functional
polymorphisms used for human identification and effect, such as the centromeres (where the chromosome
paternity testing. attaches to the spindle apparatus for proper segregation
DNA mutations can affect a single nucleotide or during cell division), which are not defined by specific
millions of nucleotides, even whole chromosomes, DNA sequences.2
and thus can be classified into three categories: gene, A eukaryotic chromosome is a double helix of DNA.
chromosome, and genome mutations. Gene muta- A cell nucleus contains 4 cm of double helix, which must
tions affect single genes and are often, but not always, be compacted, both to fit into the nucleus and to accu-
small changes in the DNA sequence. Chromosome rately segregate in mitosis. An extended DNA double
mutations affect the structures of entire chromosomes. helix undergoes an 8,000-fold compaction to make a
These changes require movement of large chromosomal metaphase chromosome (Fig. 7.1).3
regions (hundreds of thousands to millions of base The winding of DNA onto histones, the most abun-
pairs) either within the same chromosome or to another dant proteins in the cell, is the first step in compaction.
chromosome. Approximately 160 to 180 base pairs (bp) of DNA are
Genome mutations are changes in the number of wrapped around a set of eight histone proteins (two
chromosomes. A cell or cell population with a normal each of H2a, H2b, H3, and H4) to form a nucleosome.
complement of chromosomes is euploid. Genome muta- Nucleosomes are visible by electron microscopy as
tions result in cells that are aneuploid. Aneuploidy is 100-Å beadlike structures that are separated by short
mostly observed as increased numbers of chromo- (70–90 bp) strands of a free double helix or linker DNA
somes because the loss of whole chromosomes is gen- (Fig. 7.2). This “bead-on-a-string” arrangement com-
erally not compatible with survival. Aneuploidy in prises the 10-nm.
diploid organisms can result when there are more than The structure of metaphase chromosomes is main-
two copies of a single chromosome or when there are tained by more than just histones. Metaphase chromatin
multiple copies of one or more chromosomes. Down is one-third DNA, one-third histones, and one-third non-
syndrome is an example of a disease resulting from histone proteins. Nonhistone protein complexes, termed
aneuploidy, where there are three copies, or trisomy, of condensin I and condensin II, maintain mitotic chromo-
chromosome 21. some structure.3
Detection of mutations in the laboratory ranges from
direct visualization of genome and chromosomal muta- Histooricaal Higghlligghtts
tions under the microscope to molecular methods to
detect single-base changes. Methods used for detection Before 1943, histones were thought to contain
of genome and chromosomal mutations are discussed in genetic information. Their function was later
this chapter. determined to be structural. It is now known that
DNA double helix
2 nm
“Beads-on-a-string” 11 nm
form of chromatin
30-nm chromatin
fibers of packed 30 nm
nucleosomes
Chromosome in 300 nm
condensed form
Supercoiled 700 nm
chromatin fibers
FIGURE 7.1 DNA compaction into metaphase
chromosomes. Histone wrapped in DNA forms the
10-nm chromatin strands found in transcriptionally
active 10-nM DNA. Further compaction results in
1,400 nm the closed 30-nm fibers found in transcriptionally
silent DNA. (From Alberts B. Molecular biology of
the cell. 4th ed. New York, NY: Garland Science,
Duplicated chromosome 2002.)
in addition to their structural role, histones control DNA. The fibers are locally relaxed into 10-nm fibers
access to and expression of DNA. Modification of for DNA metabolism as required during the cell cycle.
histones, through acetylation, methylation, phos- When the DNA is relaxed into 10-micron fibers for tran-
phorylation, or ubiquitination, alters DNA access scription or replication, the placement of nucleosomes
and plays a role in other cellular functions, such as along the double helix can be detected using nucleases
recombination, replication, and gene expression. (e.g., Mung bean nuclease, or DNase I). These enzymes
cut the double helix in the part of the double helix that
is exposed between the histones.
In the interphase nucleus, the 10-micron fiber is further The 30-nm interphase fibers are looped onto protein
coiled around histone H1 (or H5 in certain cells) into scaffolds to form 300-nm fibers before entry into the
a thicker and shorter 30-nm or 30-micron fiber. The M phase of the cell cycle (mitosis), and the looped fibers
30-nm interphase fibers represent the “resting state” of are wound into 700-nm solenoid coils.4 The 700-nm
110 Å
H2A
H2B
H3
H4
Core DNA
55 Å
H3
H1
H2A H2B
Linker DNA
FIGURE 7.2 DNA wrapped around eight histone proteins

(two each of histones 2A, 2B, 3, and 4) forms a nucleosome. A
further association with histone H1 coils the nucleosomal DNA
into a 30-nm fiber.
FIGURE 7.3 Apoptotic DNA (A) is characterized by the
ladder seen on gel electrophoresis. The ladder forms as a result
coils are compacted into the 1,400-nm fibers that can be of nuclease activity on exposed linker regions of DNA. This is
seen microscopically in metaphase nuclei and as karyo- in contrast to randomly degraded DNA from necrotic cells (N).
types in laboratory testing.
In the 30-nm interphase chromatin fiber, the internu-
cleosomal DNA is wound into a solenoid coil. Loss of including chromosome segregation in prokaryotes.
this level of organization is the first classic indicator of Two of the SMC proteins, XCAP-C and XCAP-E,
apoptosis, or programmed cell death. The 30-nm fibers first isolated from frog eggs,5 are integral parts of
are uncoiled, and the exposed linker DNA between the condensin complex, a protein scaffold struc-
the nucleosomes becomes susceptible to digestion by ture that can be isolated from both mitotic and
intracellular nucleases. The DNA wrapped into the interphase cells. In the presence of topoisomerase,
nucleosomes remains intact so that DNA isolated from this complex can wrap DNA around itself in a
apoptotic cells contains “ladders,” or discrete multiples reaction driven by adenosine triphosphate (ATP).
of approximately 180 bp. These ladders resolved by SMC family proteins also play a role in the repair
agarose gel were one of the earlier molecular methods of chromosomal breaks.6 Although the exact role
used to detect or confirm cell death by apoptosis of the SMC proteins in chromosome condensation
(Fig. 7.3). and decondensation is not yet completely defined,
Chromosome topology (state of compaction of the this ability to change chromosome architecture is a
DNA double helix) affects gene activity. Highly com- significant feature of DNA metabolism.7
pacted DNA is less available for RNA transcription. The
more highly compacted state of DNA is closed chro-
matin, or heterochromatin (in contrast to open chroma-
tin, or euchromatin). Maintenance of heterochromatin Chromosome Morphology
throughout interphase may require condensin proteins or Mitotic chromosomes have been distinguished his-
condensin-like protein complexes (nonhistone proteins). torically by their relative size and centromere place-
ment. The centromere is the site of attachment of the
chromosome to the spindle apparatus. The connec-
Advanced Concepts tion is made between microtubules of the spindle and
a protein complex, the kinetochore, that assembles at
Members of a family of proteins called SMC pro- the centromere sequences (Fig. 7.4). At the nucleotide
teins control chromosome condensation in eukary- level, the centromere is composed of a set of highly
otes and other aspects of chromosome behavior, repetitive alpha satellite sequences.8,9 These repetitive
sequences interfere with chromosome compaction so
Centromere High-order array
Monomers (171 bp)

alpha satellite DNA
Kinetochore
Chromatin
Spindle fibers Metacentric Submetacentric Acrocentric
FIGURE 7.5 The arms of metacentric chromosomes (left) are

Inner layer Middle layer Outer layer of equal size. Submetacentric chromosomes (center) divide the
(40–60 nm) (25–30 nm) (40–60 nm) chromosome into long arms and short arms. Acrocentric cen-
tromeres (right) are very near the ends of the chromosome.
FIGURE 7.4 The centromere (top) consists of tandem repeats
of 171 base-pair sequences flanking sets of single-repeat units,
or monomers repeated in groups in a higher-order repeat array. Chromosomes 13 to 15, 21, and 22 are considered acro-
The kinetochore (bottom) is a protein structure that connects centric, but may be classified as subtelocentric.
the centromere chromatin to the spindle apparatus.
Visualizing Chromosomes
that microscopically, the centromere appears as a con-
striction in the metaphase chromosome. Chromosomes Conventional cytological stains, such as Feulgen,
are metacentric, submetacentric, acrocentric, or telo- Wright, and hematoxylin have been used to visualize
centric, depending on the placement of the centromere chromosomes. An advance in the recognition of indi-
(Fig. 7.5). The placement of the centromere divides the vidual chromosomes was the discovery that fluorescent
chromosome into arms. Metacentric chromosome arms stains and chemical dyes can react with specific chro-
are approximately equal in length, whereas one arm is mosome regions.10 This region-specific staining forms
longer than the other in submetacentric chromosomes. reproducible patterns where portions of the chromosome
One arm is extremely small or missing in acrocentric or accept or reject the stain. For cytogenetic analysis, this
telocentric chromosomes, respectively. allows unequivocal identification of every chromosome
and the direct detection of some chromosomal abnor-
malities. Underlying the region-specific staining is the
Advanced Concepts implication that the reproducible staining patterns occur
as a result of defined regional ultrastructures of the
Some plants and insects have holocentric chromo- mitotic chromosomes.
somes. During cell division, these chromosomes When chromosomes are stained with the fluorescent
associate with kinetochores along their entire dyes, quinacrine and quinacrine mustard, the result-
length. ing fluorescence pattern visualized after staining is
Q banding (Fig. 7.6). This method was first demon-
strated in 1970 by Caspersson, Zech, and Johansson.11
Human chromosomes are acrocentric or submetacentric The results of this work confirmed that each human
and so have long and short arms (Table 7.2). The long chromosome could be identified by its characteristic
arm of a chromosome is designated q, and the short arm banding pattern. Q banding gives a particularly intense
is designated p. Acrocentric chromosomes have a ratio staining of the human Y chromosome and thus may also
of long arm length:short arm length from 3:1 to 10:1. be used to distinguish the Y chromosome in interphase
TABLE 7.2 Classification of Chromosomes

by Size and Centromere Position
Group Chromosomes Description
A 1, 2 Large metacentric
3 Large submetacentric
B 4, 5 Large submetacentric Centromere
C 6–12, X Medium-sized submetacentric
D 13–15 Medium-sized acrocentric with

satellites
E 16 Short metacentric
17, 18 Short submetacentric
F 19, 20 Short metacentric
G 21, 22 Short acrocentric with satellites
Y Short acrocentric
G or Q banding R banding C banding
FIGURE 7.6 Reproducible staining patterns on chromosomes

are used for identification and site location. Heterochromatin
stains darkly by G or Q banding (left); euchromatin stains
nuclei. Because Q banding requires a fluorescent micro- darkly by R banding (center); C banding stains centromeres
scope, it is not as widely used as other stains that are (right).
detectable by light microscopy.
The chemical dye Giemsa stains in patterns, or G
bands, similar to those seen in Q banding. The appear- pattern opposite to the G banding pattern called
ance of G banding differs, depending on the treatment R banding. R bands can also be visualized after
of the chromosomes before staining. Mild treatment staining with acridine orange.15 Alkali treatment
(2× standard saline citrate for 60 minutes at 60°C) yields of chromosomes results in centromere staining, or
the region-specific banding pattern comparable to that C banding.16
seen with fluorescent dyes.
Histooricaal Higghlligghtts Centromere staining is absent in G-band patterns and

may be associated with heterochromatin, the “quiet,” or
Trypsin or other proteolytic extraction or dena- poorly transcribed, sequences along the chromosomes
turation of proteins before Giemsa staining was that are also present around centromeres. In contrast,
found to map structural aberrations more clearly euchromatin, which is relatively rich in gene activ-
and became the most commonly used staining ity, may not be stained as much as heterochromatin in
method for analyzing chromosomes.12,13 G bands C banding. The correlation between heterochromatin
were also produced by Feulgen staining after and staining may also hold for noncentromeric G and
treatment with DNase I.14 Harsher treatment of Q bands. This association is complicated, however,
chromosomes (87°C for 10 min, then cooling because a variety of procedures and stains can produce
to 70°C) before Giemsa staining will produce a identical banding patterns. The correlation of staining
with heterochromatin is contradicted by observations Chromosome 17

of the X chromosome. Although one X chromosome is Arm Region Band Subband
inactive and replicates later than the active X in females,
both X chromosomes stain with equal pattern and inten- 3
sity. Staining differences, therefore, must be due to other 2
2
factors. Possible explanations for differential interac- 2
1
2
tions with dye include differences in DNA compaction, 1
1
sequences, and DNA-associated nonhistone proteins. p 5
The number of visualized bands can be increased 1 4
1 3
from about 300 to 500 per chromosome by staining 2
chromosomes before they reach maximal metaphase 1
condensation. This is called high-resolution banding. 1
17q11.2
Nucleolar organizing region (NOR) staining is 2
1
another region-specific staining approach. Chromo- 1 3
somes treated with silver nitrate will stain specifically 1
at the constricted regions, or stalks, on the acrocentric 2 2
q 3
chromosomes.16 1
Staining of chromosomes with 4′,6-diamidino- 3 2, 3
4
2-phenylindole (DAPI) was first described in 1976 1
2
as a way to detect mycoplasmal contamination in cell 2
3
cultures.17 DAPI binds to the surface grooves of dou- 4
ble-stranded DNA and fluoresces blue under ultraviolet
(UV) light (353-nm wavelength). DAPI is used to visu-
alize chromosomes as well as whole nuclei. FIGURE 7.7 Identification of chromosomal location by
Chromosome banding facilitates the detection of G-band patterns. Locations are designated by the chromosome
deletions, insertions, inversions, and other abnormalities number 17 in this example, the arm q, the region 1, the band 1,
and the sub-band 2.
and the identification of distinct chromosomal locations.
For this purpose, the reproducible G-banding pattern has
been ordered into regions, comprising bands and sub-
bands. For example, in Figure 7.7, a site on the long arm by addition of a mitogen, usually phytohemagglutinin.
(q) of chromosome 17 is located in region 1, band 1, Dividing cells are then arrested in metaphase with Col-
sub-band 2, or 17q11.2. cemid, an inhibitor of microtubule (mitotic spindle) for-
mation. The chromosomes in dividing cells that arrest
in metaphase will yield a chromosome spread when
DETECTION OF GENOME the cell nuclei are disrupted with hypotonic buffer. The
23 pairs of chromosomes can then be assembled into an
AND CHROMOSOMAL MUTATIONS organized display, or karyotype, according to their size
and centromere placement (Fig. 7.8). Aneuploidy may
Karyotyping
be observed affecting several chromosomes18 (Fig. 7.9)
Genome mutations, or aneuploidy, can be detected by or a single chromosome (Fig. 7.10).
indirect methods, such as flow cytometry, and more Karyotyping can also be used to detect chromo-
directly by karyotyping. A karyotype is the complete set somal mutations such as translocations, which are the
of chromosomes in a cell. Karyotyping is the direct obser- exchange of genetic material between chromosomes.
vation of metaphase chromosome structure by arranging Translocations can be of several types. In reciprocal
metaphase chromosomes according to size. This requires translocations, parts of two chromosomes exchange;
collecting living cells and growing them in culture in the that is, each chromosome breaks, and the broken chro-
laboratory for 48 to 72 hours. Cell division is stimulated mosomes reassociate or recombine with one another.
FIGURE 7.8 A normal male karyotype. There are 22 pairs of autosomes, one inherited from each parent, and one pair of sex
chromosomes, XY. This karyotype is designated 46,XY.
When this type of translocation results in no gain nor

loss of chromosomal material, it is balanced (Figs. 7.11
and 7.12). Balanced translocations may occur, therefore,
1 2 3 4 5
without phenotypic effects. Balanced translocations in
germ cells (cells that give rise to eggs or sperm) can,
however, become unbalanced by not assorting properly
during meiosis; as a result, they affect the phenotype
6 7 8 9 10 11 12 of offspring. A robertsonian translocation involves the
movement of the long arm of an acrocentric chromosome
to the centromere of another acrocentric chromosome
(Fig. 7.13). This type of translocation may also become
13 14 15 16 17 18
unbalanced during reproduction, resulting in a net gain
or loss of chromosomal material in the offspring.
Other types of chromosome mutations that are visible
by karyotyping are shown in Figure 7.14. A deletion is a
19 20 21 22 X loss of chromosomal material. Large deletions covering
FIGURE 7.9 Aneuploidy involving multiple chromosomes. millions of base pairs can be detected using karyotyping;
Chromosomes 5 and 12 are show trisomy; chromosomes 6, 9, smaller microdeletions are not always easily seen using
and 16 are show monosomy. this technique. An insertion is a gain of chromosomal
FIGURE 7.10 Aneuploidy involving Y chromosome disomy (XYY syndrome). This is designated 47,XYY.
material. The inserted sequences arise from duplication Histooricaal Higghlligghtts

of particular regions within the affected chromosome or
from fragments of other chromosomes. As with dele- The first chromosome mutations associated with
tions, insertions cause altered banding patterns and a human disease were visualized in the 1960s in
change in the size of the chromosomes. Inversions leukemia cells. Peter Nowell and David Hunger-
result from excision, flipping, and reconnecting chromo- ford observed an abnormally small chromosome
somal material within the same chromosome. Pericen- 22 in leukemia cells, which was labeled the “Phil-
tric inversions include the centromere in the inverted adelphia” chromosome. A few years later, Janet
region, whereas paracentric inversions involve Rowley, using chromosome banding, noted that
sequences within one arm of the chromosome. An iso- tumor cells not only lost genetic material, but
chromosome is a metacentric chromosome that results they exchanged it. In 1972 she first described the
from transverse splitting of the centromere during cell translocation between chromosomes 8 and 21,
division. Transverse splitting causes two long arms or t(8;21) in patients with acute myeloblastic leu-
two short arms to separate into daughter cells instead of kemia. In that same year, she demonstrated that
normal chromosomes with one long arm and one short the Philadelphia chromosome was the result of a
arm. The arms of an isochromosome are therefore equal reciprocal exchange between chromosome 9 and
in length and genetically identical. A ring chromosome chromosome 22.19 She went on to identify addi-
results from deletion of genetic regions from ends of the tional reciprocal translocations in other diseases:
chromosome and a joining of the ends to form a ring. the t(14;18) translocation in follicular lymphoma
A derivative chromosome is an abnormal chromosome and the t(15;17) translocation in acute promyelo-
consisting of translocated or otherwise rearranged parts cytic leukemia. This was also the first evidence
from two or more unidentified chromosomes joined to a that cancer had a genetic basis.
normal chromosome.
FIGURE 7.13 An isochromosome. The long arms of two

FIGURE 7.11 A balanced reciprocal translocation. At the res- acrocentric chromosomes are joined at the centromere.
olution of karyotyping, no chromosomal material (banding) is
lost.
FIGURE 7.12 A karyotype showing a

balanced reciprocal translocation be-
tween chromosomes 5 and 13. This is
designated 46,XX,t(5;13).
Translocation Deletion Inversion
FIGURE 7.14 Chromosome mutations involv-

ing alterations in chromosome structure. In
addition to translocations, ring and derivative
chromosomes may or may not result in loss of
chromosomal material. Insertions without
Ring Derivative duplication of the inserted regions and deletions
Isochromosome Insertion chromosome chromosome will result in gain or loss of DNA.
Results of karyotyping analyses are expressed as the apply to fluorescent chromosome analysis (see following
number of chromosomes per nucleus (normal is 46), the discussion), which would require a motorized scanning
type of sex chromosomes (normal is XX or XY), fol- stage, automated area selection on the slide, and signal
lowed by any genetic abnormalities observed. A normal evaluation.
karyotype is 46,XX in a female or 46,XY in a male. A
karyotype showing 46,XX,del(7)(q13) denotes a dele-
Fluorescence In Situ Hybridization
tion in the long arm q of chromosome 7 at region 1,
band 3. A karyotype showing 46,XY,t(5;17)(p13.3;p13) Fluorescence in situ hybridization (FISH) is a method
denotes a translocation t between the short arms p of widely used to detect protein and RNA as well as DNA
chromosomes 5 and 17 and region 1, band 3, sub- structures in place in the cell, or in situ.20 FISH offers a
band 3, and region 1, band 3, respectively. A karyotype more rapid assay with higher resolution and flexibility
showing 47,XX+21 is the karyotype of a female with than karyotyping.21 FISH targets specific sequences of
Down syndrome resulting from an extra chromosome chromosomes with fluorescent probes. Even though FISH
21. Klinefelter syndrome is caused by an extra X chro- offers higher resolution than karyotyping for specific
mosome in males, for example, 47,XXY. Table 7.3 lists targets, it is limited to the regions complementary to the
some of the terms used in expressing karyotypes. FISH probes. Probes are designed to hybridize to critical
Manual assembly of karyotypes from microscopic areas that are amplified, deleted, translocated, or other-
images has been replaced by software systems that elec- wise rearranged in disease states. Unlike karyotyping
tronically arrange the chromosomes from the image of that is performed under a light microscope, FISH anal-
the chromosome spread. Although this automation is ysis requires a fluorescence microscope that will excite
highly applicable to the static images of chromosome fluorescent emission for the probes and special filters
spreads, such an automated system is more difficult to for detection of fluors emitting at different wavelengths.
Probes hybridized
TABLE 7.3 A List of Descriptive Abbreviations Cell nucleus to chromosomes
Abbreviation Indication
+ Gain
− Loss
del Deletion
Normal cell (diploid) Triploid Deletion
der Derivative chromosome
FIGURE 7.15 FISH analysis using centromeric probes for a
dup Duplication normal diploid cell (left), triploidy or trisomy (center), and
deletion or monosomy (right).
ins Insertion
inv Inversion
correlate with the state of that chromosome or locus.
i, iso Isochromosome For example, a probe to any unique region on chro-
mosome 22 should yield an image of two signals per
mat Maternal origin
nucleus, reflecting the two copies of chromosome 22 in
pat Paternal origin the somatic cell nucleus (Fig. 7.15). A deletion or dupli-
cation of the DNA that is hybridized to the probe will
r Ring chromosome
result in a nucleus with only one signal or more than
t Translocation two signals, respectively. Multiple probes spanning large
regions are used to detect regional deletions.22
tel Telomere (end of chromosome arm)
Translocations or other rearrangements are detected
using probes of different “colors” (or signals) comple-
mentary to regions on each chromosome taking part in
the translocation. A normal nucleus will have two of
Analysis of signals from FISH also requires expertise in
each of the probe signals. A translocated chromosome
reading signals in three dimensions.
will combine two of the probe signals, resulting in a loss
of one of each signal in the nucleus. Analysis of translo-
Interphase FISH
cation signals is sometimes complicated by false signals
In contrast to karyotyping, interphase FISH does not that result from two chromosomes landing close to one
require culturing of cells. Because growing cells in another in the nucleus, such that the bound probes give a
culture is not required, interphase FISH methods are signal similar to that exhibited by a translocation. These
used commonly to study prenatal samples, tumors, and false signals may be distinguished from true translocations
hematological malignancies, not all of which are conve- by the size of the fluorescent image, or by vertical focus-
niently brought into metaphase in culture. ing with the microscope. Accounting for false-positive
For FISH cytogenetic analysis, fixed cells are perme- signals as background noise limits the sensitivity of
abilized and exposed to a probe. The probe is a 60- to this assay.
200-kb fragment of DNA attached covalently to a fluo- The sensitivity of interphase FISH analysis for the
rescent molecule. The probe will hybridize, or bind, to detection of translocations is increased through the use
its complementary sequences in the cellular DNA. In of dual-color probes, or dual-fusion probes. These
interphase FISH, the bound probe is visualized under probes are mixtures of two single probes, each labeled
a fluorescent microscope as a point of fluorescent light with a different fluorescent dye. They are designed to
in the nucleus of the cell. Probes are designed to be bind to regions spanning the breakpoint of transloca-
complementary to a particular chromosome or chromo- tions. A translocation will be observed as a signal from
somal locus so that the image under the microscope will both the translocation junction and the reciprocal of the
Cell nucleus Breakpoint
Probes
Probes
Normal Translocation
Translocated
chromosome FIGURE 7.17 Break-apart probes bind to the chromosome
flanking the translocation breakpoint region. Normal cells will
display the combination signal (bottom left), and a transloca-
tion will separate the probe signals (bottom right).
surrounding centromeres. These probes detect aneusomy

Reciprocal of any chromosome. Combinations of centromeric probes
translocation
and region-specific probes are often used to confirm
Translocated product
deletions or amplifications in specific chromosomes.
chromosome
Addition of a CEN probe to dual-color probes comprises
FIGURE 7.16 FISH analysis using distinct probes to detect a a tricolor probe and serves as a control for amplification
translocation. A normal nucleus has two signals from each or loss of one of the chromosomes involved in the trans-
probe (top). A translocation involving the two chromosomes location. For example, the IGH/MYC/CEP 8 Tri-color
combines the two probe colors (middle). Dual-fusion probes probes are a mixture of a 1.5-Mb–labeled probe, com-
confirm the presence of the translocation by also giving a plementary to the immunoglobulin heavy-chain region
signal from the reciprocal breakpoint (bottom). See Color
(IGH) of chromosome 14; an approximately 750-kb dis-
Plate 2.
tinctly labeled probe complementary to the myc gene on
chromosome 8; and a CEN to chromosome 8.
Each chromosome arm has a unique set of repeat
translocation junction, for example, t(9;22) and t(22;9) sequences located just before the end of the chromo-
(Fig. 7.16). Dual-color break-apart probes, 0.6 to some (the telomere; Fig. 7.18). These sequences have
1.5 Mb, are another approach to decrease background been studied to develop a set of DNA probes specific
signals as well as to identify translocation events where to the telomeres of all human chromosomes. Telomeric
one chromosome can recombine with multiple poten- probes are useful for the detection of chromosome
tial partners. These probes are designed to bind to the structural abnormalities, such as cryptic translocations
intact chromosome flanking the translocation break- or sub-telomeric deletions that are not easily visualized
point. When a translocation occurs, the two probes sep- by standard karyotyping.
arate (Fig. 7.17). Sometimes called tri-FISH, break-apart Because interphase cells for FISH do not require cul-
probes are not the same as tricolor probes (see following turing of the cells and stimulating division, it is valuable
discussion). for analysis of cells that do not divide well in culture,
Centromeric probes (CEN probes) are designed to including fixed cells. Furthermore, because hundreds
hybridize to highly repetitive alpha satellite sequences of cells are analyzed microscopically using FISH, the
Probe-binding site Telomere
FIGURE 7.18 The binding sites for telomeric 100–200 kb 3–20 kb

probes are unique sequences just next to the telo- Unique sequences Telomere-
meric associated repeats and telomeric repeat associated repeats
sequences at the ends of chromosomes. (TTAGGG)n
sensitivity of detection is higher than that of metaphase sequences to reduce nonspecific binding) or facilitators
procedures, which commonly examine 20 spreads. A such as dextran sulfate (to increase the effective probe
limitation of FISH, however, is the inability to identify concentration) depends on the sequence complexity of
chromosomal changes other than those at the specific the probe. One to 10 micrograms of probe may be used
binding region of the probe(s). In contrast, karyotyp- in a hybridization volume of 3 to 10 μL. The hybrid-
ing, a more generic method, can detect any chromo- ization of the probe on the target cells is performed at
somal change that causes changes in chromosomal size, 37°C to 42°C in a humidified chamber. The slides are
number, or banding pattern within the sensitivity limits cover-slipped and sealed to optimize the hybridization
of the procedure. conditions.
Preparation of the sample is critical in interphase Following hybridization and rinsing off of the
FISH analysis, both to permeabilize the cells for optimal unbound probe, the sample is observed microscopically.
probe–target interaction and to maintain cell morphol- The probe signals should be visible from entire intact
ogy.23 Optimal results are obtained if fresh interphase nuclei. Adequate numbers of cells must be visible, but
cells are incubated overnight (aging) after deposition crowded cells where the nuclei and signals overlap do
on slides. After aging overnight, cells are treated with not yield accurate results. Furthermore, different tissue
protease to minimize interference from cytoplasmic types have different image qualities and characteristics
proteins and fixed with 1% formaldehyde to stabilize that must also be taken into account when assessing the
the nuclear morphology. Before DNA denaturation, the FISH image. Another complication of FISH analysis is
cells are dehydrated in graded concentrations of ethanol. photobleaching (fading) or loss of probe signal emis-
Paraffin-embedded fixed tissues are dewaxed in xylene sion (105 photons/second) due to photochemical destruc-
before protease and formaldehyde treatment. tion of the fluorophore molecules. For this reason,
The quality of the probe also has to be checked and FISH slides should not be subjected to prolonged light
its performance validated before use. Fluorescent probes exposure.
(DNA with covalently attached fluorescent dyes) are
usually purchased from vendors, which may also supply
Metaphase FISH
compatible hybridization reagents and controls. Never-
theless, the probe performance should be observed on Metaphase analysis has been enhanced by the devel-
control tissue before use on patient samples. Under a flu- opment of fluorescent probes that bind to metaphase
orescent microscope with the appropriate color-distinc- chromosomal regions or to whole chromosomes. Meta-
tion filters, the signal from the probe should be bright, phase FISH allows analysis of small regions not visible
specific to the target in the cell nuclei, and free of high by regular chromosome banding. Probes that cover the
background noise. Probes differ in their signal charac- entire chromosome, or whole chromosome paints,
teristics and intensities; the technologist should become are valuable for detecting these small or complex rear-
familiar with what to expect from a given probe on dif- rangements (Fig. 7.19). By mixing combinations of five
ferent types of tissues. This is facilitated by comparison fluors and using special imaging software, spectral
with controls accompanying each run of samples. karyotyping can distinguish all 23 chromosomes by
As in Southern and northern blotting procedures, chromosome-specific colors.24 This type of analysis can
both probe and target must be denatured prior to hybrid- detect abnormalities that affect multiple chromosomes,
ization. The amount of time taken to hybridize and use as is sometimes found in cancer cells or immortalized
Cot-1 DNA (placental DNA enriched for repetitive cell lines.25,26 Telomeric and centromeric probes are also
FIGURE 7.21 Multicolor FISH analysis simultaneously

reveals structural or numerical abnormalities in three loci. See
Color Plate 4.
acetic acid may improve the chromosome spreading and

decrease background noise. Condensed chromosome
spreads, especially those from cultured metaphases, may
be affected by temperature and humidity. Under a phase
FIGURE 7.19 Chromosome painting showing a derivative
contrast microscope, the chromosomes should appear
chromosome formed by movement of a fragment of chromo-
some 12 (black) to an unidentified chromosome. See Color well separated with sharp borders. Cytoplasm should
Plate 3. not be visible. Once the slide is dried, hybridization pro-
ceeds as discussed previously for interphase FISH.
Combinations of different locus-specific probes and
chromosome paints can be used simultaneously, yield-
ing more information than carrying out separate experi-
ments on multiple specimens. The generic term for this
is multicolor FISH (QMFISH or M-FISH). Imaging
with 10 to 20 different probes or with a combination
of region-specific probes and spectral karyotyping dif-
ferentiates multiple chromosomes by spectral properties
(Fig. 7.21). Simultaneously, M-FISH identifies specific
chromosomal regions based on the presence or absence
of the probe color visualized with specific filters. Anal-
ysis by both may show cryptic translocations and inser-
Centromeric probes Telomeric probes tions as well as the chromosomal origins of marker
FIGURE 7.20 Centromeric (left) and telomeric (right) probes chromosomes.27
on metaphase chromosomes.
Advanced Concepts
applied to metaphase chromosomes (Fig. 7.20) to detect Quantitative FISH (Q-FISH; Fig. 7.22) is the anal-
aneuploidy and other genomic mutations. ysis of repeated sequences by assessing the relative
Preparation of chromosomes for metaphase FISH intensity of probe signals. Microscopic images are
procedures begins with the culture of cells for 72 hours. digitized on a charge-coupled device (CCD).28,29
About 45 minutes before harvesting, colcemid is added The signals are then measured by imaging soft-
to the cultures to arrest dividing cells in metaphase. ware, quantifying FISH signals from each digital
The cells are then suspended in a hypotonic medium image. The relative intensity is compared between
(0.075 M KCl) and fixed with methanol/acetic acid signals. The telomere Q-FISH technique has been
(3:1). The fixed-cell suspension is applied to an inclined applied to studies on telomere length.30
slide and allowed to dry. A second treatment with 70%
Normal reference DNA
Test sample DNA
Amplification
Deletion
FIGURE 7.23 In CGH, the test sample is compared with a

normal reference sample on a metaphase spread. Normally, test
and reference signals are equal. A higher test signal denotes an
amplification, and a higher reference signal denotes a deletion.
FIGURE 7.22 Fluorescent signals are digitized and quanti- the far-red region of the spectrum (650 to 667 nm), is
fied in quantitative FISH. The relative intensity of signals in represented as “red.” Derivatives of these dyes, such as
multiple loci will demonstrate copy-number variations. Cy3.5, which fluoresces in the red-orange region, are
also available. Because these dyes fluoresce brightly and
are water-soluble, they have been used extensively for
CGH using imaging equipment.
Labeling (attachment of Cy3 or Cy5 dye to the test
and reference DNA) is achieved by nick translation or
primer extension in which nucleotides covalently at-
COMPARATIVE GENOME HYBRIDIZATION (CGH) tached to the dye molecules are incorporated into the
DNA sequences. The dye nucleotides commonly used
Intrachromosomal amplifications or deletions can be de- for this type of labeling are 5-amino-propargyl-2′-
tected by comparative genome hybridization (CGH).31 deoxycytidine 5′-triphosphate coupled to the Cy3 or
In this method, DNA from test and reference samples Cy5 fluorescent dye (Cy3-AP3-dCTP, Cy5-AP3-dCTP)
is labeled and used as a probe on a normal metaphase or 5-amino-propargyl-2′-deoxyuridine 5′-triphosphate
chromosome spread (Fig. 7.23). One advantage of CGH coupled to the Cy3 or Cy5 fluorescent dye (Cy3-AP3-
is its capability to identify the location of deletions or dUTP, Cy5-AP3-dUTP). Separate aliquots of test and
amplifications throughout the genome (Fig. 7.24); the reference DNA are labeled with different Cy3 and Cy5
resolution (precise identification of the amplified or de- dyes, respectively, before application to a normal meta-
leted region), however, is not as high as can be achieved phase spread. Test DNA is partially digested with DNase
with array CGH.32,33 to produce fragments that will bind efficiently to the de-
For CGH, the test DNA is isolated and labeled along natured DNA in a metaphase chromosome spread. Al-
with a reference DNA. Two colorimetrically distinct though CGH requires advanced technical expertise, it
cyanine dyes, commonly Cy3 and Cy5, are used as flu- has shown value in identifying recurrent genomic im-
orescent labels for the test and reference DNA. Cy3, balances not detected by karyotyping. Future advances
which fluoresces at a wavelength of 550 nm, is often and incorporation of high-density arrays will increase
represented as “green,” and Cy5, which fluoresces in the ease and scope of use of this technology.
0.5 1.0 2.0
(Amplified region)
0.5 1.0 2.0

FIGURE 7.24 CGH analysis of four chromo-
somes of cells from a cancer cell line. Ampli-
fied or deleted areas are observed where the
test and reference signals are not equal. The
vertical lines in the center of the diagram rep-
(Deleted region) resent the ratio of test/reference signals on the
chromosomal spread showing excess test or
excess reference signal (right idiograms).
STUDY QUESTIONS 47,XY, +18

46,XY, del(16)p(14)
1. What chromosomal location is indicated by iso(Xq)
15q21.1? 46,XX del(22)q(11.2)
45,X
2. During interphase FISH analysis for the t(9;22)
translocation, one nucleus was observed with two 5. A chromosome with a centromere located such
normal signals (one red for chromosome 22 and that one arm of the chromosome is longer than the
one green for chromosome 9) and one composite other arm is called
red/green signal. Five hundred other nuclei
a. metacentric.
were normal. What is one explanation for this
b. paracentric.
observation?
c. telocentric.
d. submetacentric.
3. Is 47;XYY a normal karyotype?
4. Write the numerical and structural chromosomal 6. A small portion from the end of chromosome 2
abnormalities represented by the following has been found on the end of chromosome 15,
genotypes: replacing the end of chromosome 15, which has
moved to the end of chromosome 2. This mutation metaphase FISH, is better for accurate detection of
is called a(n) this abnormality? Why?
a. reciprocal translocation.
13. The results of a CGH analysis of Cy3 (green)-
b. inversion.
labeled test DNA with Cy5 (red)-labeled reference
c. deletion.
DNA on a normal chromosome spread revealed
d. robertsonian translocation.
a bright red signal along the short arm of
chromosome 3. How is this interpreted?
7. Phytohemagglutinin is added to a cell culture when
preparing cells for karyotyping. The purpose of the a. 3p deletion
phytohemagglutinin treatment is to b. 3q deletion
c. 3p amplification
a. arrest the cell in metaphase.
d. 3q amplification
b. spread out the chromosomes.
c. fix the chromosomes on the slide.
14. A break-apart probe is used to detect a
d. stimulate mitosis in the cells.
translocation. The results of FISH analysis show
two signals in 70% of the nuclei counted and
8. A centromeric probe is used to visualize
three signals in 30% of the nuclei. Is there a
chromosome 21. Three fluorescent signals are
translocation present?
observed in the cell nuclei when stained with
this probe. These results would be interpreted as
15. What FISH technique is most useful for the
consistent with
detection of multiple complex genomic mutations?
a. a normal karyotype.
b. Down syndrome.
c. Klinefelter syndrome. References
d. technical error.
1. Murray A. How to compact DNA. Science 1998;282:425–427.
2. Black B, Foltz DR, Chakravarthy S, Luger K, Woods VL, Cleve-
9. Cells were harvested from a patient’s blood, land DW. Structural determinants for generating centromeric chro-
cultured to obtain chromosomes in metaphase, fixed matin. Nature 2004;430:578–582.
onto a slide, treated with trypsin, and then stained 3. Piskadlo E, Oliveira RA. Novel insights into mitotic chromosome
with Giemsa. The resulting banding pattern is called condensation. F1000Research 2016;5:25.
4. Porter I, Khoudoli GA, Swedlow JR. Chromosome condensa-
a. G banding. tion: DNA compaction in real time. Current Biology 2004;14:
b. Q banding. R554–R556.
c. R banding. 5. Hirano T, Mitchison TJ. A heterodimeric coiled-coil protein required
for mitotic chromosome condensation in vitro. Cell 1994;79:
d. C banding. 449–458.
6. Kinoshita E, van der Linden E, Sanchez H, Wyman C. RAD50,
10. A FISH test with a centromere 13 probe is ordered an SMC family member with multiple roles in DNA break repair:
for a suspected case of Patau syndrome (trisomy how does ATP affect function? Chromosome Research 2009;17:
13). How many signals per nucleus will result if 277–288.
7. Hudson D, Ohta S, Freisinger T, Macisaac F, Sennels L, Alves F,
the test is positive for Patau syndrome? Lai F, Kerr A, Rappsilber J, Earnshaw WC. Molecular and genetic
analysis of condensin function in vertebrate cells. Molecular and
11. What would be the results if a centromere Cellular Biology 2008;19:3070–3079.
13 probe was used on a case of Edward syndrome 8. Sullivan L, Chew K, Sullivan BA. Alpha satellite DNA variation
(trisomy 18)? and function of the human centromere. Nucleus 2017;13:331–339.
9. Waye J, Willard HF. Chromosome-specific alpha satellite DNA:
nucleotide sequence analysis of the 2.0 kilobasepair repeat from
12. Angelman syndrome is caused by a microdeletion the human X chromosome. Nucleic Acids Research 1985;13:
in chromosome 15. Which method, karyotyping or 2731–2743.
10. Huang H, Chen J. Chromosome bandings. Methods in Molecular 24. Macville M, Veldman T, Padilla-Nash H, Wangsa D, O’Brien P,
Biology 2017;1541:59–66. Schrack E, Ried T. Spectral karyotyping, a 24-colour FISH tech-
11. Caspersson T, Zech L, Johansson C. Differential banding of nique for the identification of chromosomal rearrangements. His-
alkylating fluorochromes in human chromosomes. Experimental tochemistry and Cell Biology 1997;108:299–305.
Cell Research 1970;60:315–319. 25. Liang J, Ning Y, Wang R, Padilla-Nash HM, Schrock E, Soenksen
12. Seabright M. A rapid banding technique for human chromosomes. D, Nagarajan L, Ried T. Spectral karyotypic study of the HL-60
Lancet 1971;2:971–972. cell line: detection of complex rearrangements involving chromo-
13. Seabright M. The use of proteolytic enzymes for the mapping of somes 5, 7, and 16 and delineation of critical region of deletion
structural rearrangements in the chromosomes of man. Chromo- on 5q31.1. Cancer Genetics and Cytogenetics 1999;113:105–109.
soma 1972;36:204–210. 26. Mehra S, Messner H, Minden M, Chaganti RS. Molecular cytoge-
14. Burkholder G, Weaver M. DNA protein interactions and chromo- netic characterization of non-Hodgkin lymphoma cell lines. Genes
some binding. Experimental Cell Research 1977;110:251–262. Chromosomes Cancer 2002;33:225–234.
15. Bobrow M, Madan, K. The effects of various banding procedures 27. Bayani J, Squire J. Multi-color FISH techniques. Current Proto-
on human chromosomes studied with acridine orange. Cytogenet- cols in Cell Biology 2004;22.
ics and Cell Genetics 1973;12:145–156. 28. Iourov I, Soloviev IV, Vorsanova SG, Monakhov VV, Yurov
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chromosomes. Cytogenetics 1971;10:81–86. in situ hybridization (FISH) signals for applied human molecu-
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DAPI for the detection of mycoplasma in cell cultures. Medycyna 2005;53:401–408.
doświadczalna i mikrobiologia 1976;28:161–173. 29. Iourov I. Quantitative fluorescence in situ hybridization (QFISH).
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19. Mayall B, Carrano AV, Moore DH, Rowley JD. Quantification by Shimomura N, Nakamura K, Ishikawa N, Poon SS, Fujiwara M,
DNA-based cytophotometry of the 9q+/22q-chromosomal trans- Tomita K, Hiraishi N, Kuroiwa M, Matsuura M, Sanada Y, Kawano
location associated with chronic myelogenous leukemia. Cancer Y, Arai T, Takubo K. Quantitative fluorescence in situ hybridiza-
Research 1977;37:3590–3593. tion measurement of telomere length in skin with/without sun
20. Hu L, Ru K, Zhang L, Huang Y, Zhu X, Liu H, Zetterberg A, exposure or actinic keratosis. Human Pathology 2014;45:473–480.
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increasingly demanded tool for biomarker research and personal- Tachdjian G. Comparative genomic hybridization: technical
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Chapter 8
Gene Mutations
Outline Objectives
TYPES OF GENE MUTATIONS 8.1 Compare the phenotypic consequences of different
DETECTION OF GENE MUTATIONS types of point mutations.
Biochemical Methods 8.2 Distinguish the detection of known mutations from
Enzyme Immunoassays scanning for unknown mutations.
Immunohistochemistry 8.3 Explain the use of immunoassays for detecting
High-Performance Liquid Chromatography (HPLC) gene mutations and altered proteins.
Gas Chromatography 8.4 Explain the concept of separation and detection by
Mass Spectrometry HPLC, chromatography, and spectrometry.
Nucleic Acid Analyses 8.5 List and describe hybridization analyses to detect
Hybridization-Based Methods DNA mutations.
Sequencing (Polymerization)-Based Methods 8.6 Describe enzymatic cleavage methods for DNA
Enzymatic and Chemical Cleavage Methods sequence analysis.
Other Methods 8.7 Determine which detection methods are
GENE VARIANT NOMENCLATURE appropriate for the screening of new mutations or
GENE NAMES the detection of previously identified mutations.
8.8 Describe gene mutation nomenclature for
expressing sequence changes at the DNA, RNA, and
protein levels.
199
Gene mutations include deletions, insertions, inversions, concept for interpreting the results of mutation analyses.
translocations, and other changes that can affect one Substitution of one nucleotide with a different nucleo-
base pair to hundreds or thousands of base pairs. Large tide may be silent, that is, not change the amino acid
differences in DNA sequence will likely have a signifi- sequence (Table 8.1). Conservative substitutions may
cant effect on protein sequence. Alterations of a single change the amino acid sequence, but the replacement
or a few base pairs, or point mutations, will have a and the original amino acid have similar biochemical
range of effects on protein sequence. A difference of one properties (e.g., leucine for valine), and the change will
or a few base pairs may or may not change the encoded not drastically affect protein function. In contrast, a non-
amino acid. conservative substitution results in the replacement of
Point mutations are increasingly analyzed by sequenc- an amino acid with a biochemically different amino acid
ing methods. Sequencing not only directly detects the (e.g., proline for glutamine), which changes the bio-
mutated base or bases but also provides the context chemical nature of the protein. A nonsense substitution
of neighboring bases. Some sequencing methods also mutation terminates proteins prematurely when a nucle-
provide the percentage of variant alleles compared to a otide substitution produces a stop codon instead of an
reference or normal sequence. Different sequencing tech- amino acid codon. About 11% of disease-related gene
nologies have limitations with respect to certain types lesions are nonsense mutations.1
of mutations, such as structural chromosomal abnor- Insertion or deletion of other than a multiple of three
malities and copy-number variations. Next-generation nucleotides results in a frameshift mutation, throwing the
sequencing (NGS) detection of germline variants in triplet code out of frame. The amino acids in the chain
genetic testing also requires confirmation of the variant after the frameshift mutation are affected because the
by Sanger sequencing or by other means. Phenotypic triplet code will include new combinations of three nucle-
alterations in protein structure can only be predicted otides. The genetic code is structured such that frameshifts
from the nucleotide sequence. Frequently occurring vari- often terminate protein synthesis prematurely because
ants may be easily analyzed by simple and inexpensive a stop codon appears sooner in the out-of-frame coding
single-mutation tests. Therefore, standard biochemical, sequence than it would in an in-phase reading frame.
cytogenetic, and molecular methods are still important. Nonconservative, nonsense, and frameshift mutations
will generate different phenotypes, depending on where
they occur along the protein sequence.2 Point mutations
TYPES OF GENE MUTATIONS in the 3′ end of a coding region may have minimal
consequences, whereas mutations at the beginning of
Because there is more than one codon for most of the a coding sequence (5′ end or amino terminal end of a
amino acids, DNA sequence changes do not necessarily protein) are more likely to result in drastic alterations
change the amino acid sequence. This is an important or even effective deletion of the protein-coding region.
TABLE 8.1 Types of Point Mutations
DNA Sequence Amino Acid Sequence Type of Mutation
ATG CAG GTG ACC TCA GTG MQVTSV None
ATG CAG GT T ACC TCA GTG MQVTSV Silent
ATG CAA GTG ACC TCA GTG MQLTSV Conservative
ATG CCG GTG ACC TCA GTG MPVTSV Nonconservative
ATG CAG GTG ACC TGA GTG M Q V T ter Nonsense
ATG CAG GTG AAC CTC AGT G M Q V NLS Frameshift

Chapter 8 • Gene Mutations 201
These factors are important when interpreting the methods provide a more direct analysis of affected pro-
results of mutation analyses. Merely finding a change in teins and their structure and function.
a test DNA sequence does not guarantee an altered phe-
notype. Furthermore, point mutations detected by NGS Biochemical Methods
methods over large sequence regions have to be screened
to distinguish among silent, conservative, and noncon- Biochemical methods are used to directly analyze the
servative changes. For inherited or recurring diseases, change in protein structure or function rather than to
the specific change in the DNA may be ascertained from search for potential point mutations. This type of testing is
a family history or previous analysis. also useful for metabolic defects where several genes are
involved in the disease phenotype and the actual protein
or amino acid alterations can be detected using biochem-
DETECTION OF GENE MUTATIONS ical methods. Commonly used biochemical methods
include immunoassays that detect the presence of hor-
Some, mostly inherited, disease-associated sequence mones, drugs, antibodies, cancer biomarkers, and other
changes in DNA occur frequently at the same genetic metabolites in blood, urine, or other biological fluids.
location, for example, for the factor V Leiden mutation Immunohistochemistry (IHC) is a longstanding method
and the hemochromatosis C282Y, H63D, and S65C muta- that allows detection of protein abnormalities in situ so
tions. Also, increasing numbers of specific single-nucle- that the tissue and intracellular location of mutant proteins
otide polymorphisms (SNPs) are being mapped close to (or lack thereof) can be observed. In addition, automated
disease genes. These changes, although outside of the methods based on high-performance liquid or gas chro-
disease gene, are detected as specific sequence changes matography (GC) and mass spectrometry (MS) are also
frequently inherited along with the disease phenotype. frequently used; these are described later in the chapter.
Enzyme Immunoassays
Advanced Concepts Immunoassays are flexible, potentially high-through-
put methods for detecting a variety of analytes. These
The nature of the genetic code is such that frame-
methods involve the use of specific antibodies or other
shift mutations lead to a termination codon within
ligands to detect the presence of the target molecules
a small number of codons. This characteristic
(Fig. 8.1). For this type of assay, plate wells, strips, or
might have evolved to protect cells from making
capillaries coated with capture antibody are exposed to
long nonsense proteins. It is a useful parameter to
test fluid (serum, plasma, or urine) that is diluted in the
identify open reading frames in DNA.
appropriate assay buffer. If the analyte is present in the
test fluid, it will bind to the immobilized antibody. After
Some diseases are associated with many possible muta- rinsing away unbound material, detection antibodies
tions in a single gene. For instance, there are more than covalently linked to alkaline phosphatase or horseradish
600 disease-associated mutations in the cystic fibrosis peroxidase are introduced. The unbound antibody con-
transmembrane regulator (CFTR) gene, and more than jugates are washed away, and a substrate is added that
2,000 cancer susceptibility mutations have been reported will generate chemiluminescence, fluorescence, or color
in the BRCA1 and BRCA2 genes. Furthermore, unknown signals. Variations of this assay include immobilization
numbers of gene mutations are yet to be discovered. of the antigen to detect target antibodies in the test fluid.
Detection of mutations in large genes requires screen- The antibodies are then detected using anti-human anti-
ing across thousands of base pairs to detect a single body conjugates. This test is useful in the detection of
altered nucleotide. Advances in sequencing technology antibodies against infectious agents.
allow genome-wide scanning for yet-unreported muta- Enzyme immunoassay (EIA) liquid handling tech-
tions. Whole-genome sequencing, however, is currently a niques have been effectively automated. Turbidimetry
complex approach, especially with regard to interpretation. (latex agglutination), chemiluminescence, and magnetic
In some cases, biochemical-based methods are used particle methods are supported on EIA analyzers. Most
to detect or quantify the altered protein product. These work with the 96-well plate format, and there are
AP AP AP AP AP AP
FIGURE 8.1 Enzyme immunoassay formats for direct antigen detection. Antibody specific for the target analyte is immobilized
in plate wells. If present, antigen binds to the antibody and is detected with a secondary antibody conjugated to enzyme (AP, alka-
line phosphatase).
preoptimized reagent sets designed for a wide variety of was described by Coons.5 Thirty-four years later,
analytes. Taylor and Burns performed IHC on routinely
processed fixed tissues. Originally performed
Histooricaal Higghlligghtts using antisera containing polyclonal antibodies
from a natural reaction against antigen, IHC was
EIAs and enzyme-linked immunosorbent assays improved with the use of monoclonal antibodies.
(ELISAs) are modifications of an earlier tech- Monoclonal antibodies (mAbs) are produced by
nique called radioimmunoassay (RIA) that was fusion of a single antibody-producing cell with
first reported as a method to detect insulin in an immortal cell to replicate and produce many
plasma.3 A significant concern for RIA and its copies of the same antibody. Köhler and Milstein
later variations was the radioactive hazards from developed this hybridoma technique in 1975.6
iodine 131 (131I), even when it was replaced with Unlike polyclonal antibodies that target multiple
less toxic forms. In 1970, chemical methods were epitopes with varying specificity, use of mAbs
designed that coupled enzymes (alkaline phos- resulted in less nonspecific staining and better
phatase and others) with the antibodies to the image quality.
analyte. These enzymes would then produce a
signal from a color or light-producing substrate.
In 1971, Engvall and Perlmann reported the use Immunohistochemistry (IHC) is performed on thin (<5
of ELISA to detect immunoglobulin G in rabbit micron) slices of fixed or 5- to 15-micron slices of
serum.4 That same year, van Weemen and Schurs frozen tissue. Fixation of tissue in formalin preserves
demonstrated the use of EIA to quantify human tissue morphology. Sections from fixed tissue embed-
chorionic gonadotropin concentrations in urine. ded in paraffin are made on a microtome. Fixation can
also affect tissue antigens, however, altering or cover-
ing some epitopes (targets for mAb binding). Enzyme
digestion with protein-digesting enzymes (proteinase
Immunohistochemistry K, trypsin, chymotrypsin, pepsin, pronase) or heating
tissue sections in water or buffer can uncover antigen
Histooricaal Higghlligghtts epitopes. This treatment is called antigen retrieval. To
avoid the effects of formalin fixation, snap frozen tissue
Paul Ehrlich coined the term “antibody” in 1891. may be used. Tissue quickly frozen in isopentane (liquid
In 1940 immunofluorescence staining on frozen at −160°C) can be sectioned in a cryostat, which allows
sections based on antigen–antibody interactions cutting of frozen tissue into 4- to 15-micron sections
inside of a chamber held at −20°C. Frozen sections can fluorescent molecules (fluorescein, Cy5, phycoerythrin)
then be fixed in acetone, rather than formalin, to preserve attached to the bound antibodies will emit signals when
structural morphology as well as the target epitopes. The the fluor is excited. For colorimetric signals, a substrate
sections are then dried and stored frozen. Dried sections solution is added to the bound antibodies. The substrate
can be rehydrated in phosphate-buffered saline, which is oxidized by horseradish peroxidase or alkaline phos-
also will remove the embedding material used to hold phatase, leading to a color reaction. Color generation
and position the tissue in the cryostat. will depend on the substrate; however, red or brown IHC
Substances such as endogenous peroxidase, fluo- staining is the most frequently used. Staining with more
rescence, or nonspecific antibodies in the tissue may than one color may be achieved using sequential stain-
interfere with IHC results. Blocking the background ing with different antibodies and substrates.
staining with hydrogen peroxide, ultraviolet (UV) light, For direct antibody staining, the fluors or enzymes
or 1% serum will prevent background signals. Frozen are attached to the antibody that directly binds the target
sections can be treated with 0.1% sodium azide with molecules in the tissue (primary antibody). This method
0.3% hydrogen peroxide. A blocking solution contain- is faster than the alternative indirect staining, but it has
ing serum protein (albumin), detergent (Tween 20), and limited signal intensity. In indirect staining, the primary
unlabeled antibodies will minimize nonspecific binding. antibody is not attached to the signaling molecule. A
Positive and negative controls are included with samples second or secondary antibody binds the primary anti-
to ensure the adequacy and specificity of staining. body (usually from a different species, such as rabbit,
Imaging or microscopic observation of antibody mouse, or goat), carries the signal. Indirect staining may
binding requires a signal from the antibody. This signal require additional blocking with unlabeled antibodies or
can be fluorescent or colorimetric. To generate the signal, IgG protein to avoid nonspecific binding to the second-
antibodies are covalently attached to fluorescent mol- ary antibodies. The binding of a single primary antibody
ecules or enzymes (horseradish peroxidase or calf intes- by multiple secondary antibodies amplifies the signal,
tine alkaline phosphatase). For fluorescent signals, the allowing greater sensitivity of detection (Fig. 8.2).
AP AP AP AP AP AP
AP AP
(IgM)
B
FIGURE 8.2 For antibody detection, antigen is immobilized and bound to specific antibody, if present, which is detected with
AP-conjugated secondary antibody (A). Antigen may also be detected with immobilized antibodies to IgM or IgG, which will
capture antigen for detection with enzyme conjugate (B).
Staining antibodies are diluted from 100 to several Stationary phases of HPLC differ depending on the
hundred-fold or more in blocking solution. The first application. Size-exclusion columns are comprised of
blocking solution (without staining antibodies) is porous beads that exclude larger molecules and retain
removed, and the staining solution is applied to the tissue smaller molecules inside of the beads so that the larger
for 10 to 30 minutes. Stained sections are rinsed with a molecules are eluted faster than the smaller molecules
buffer (e.g., Tris-buffered saline with 0.05% Tween 20) (Fig. 8.3). Normal and reverse-phase columns sepa-
and visualized microscopically. For dual staining, the rate on the basis of hydrophilicity, with lipophilic mol-
blocking, staining, and washing procedure is repeated ecules eluting faster, or hydrophobicity, with lipophilic
with a second antibody. Visualization of fluorescent molecules eluting slower. In ion exchange, ions in the
signals is direct, but for color developed through oxida- sample are retained as counter ions to charged groups
tion of a substrate, the substrate solution is placed on the that are permanently attached to the stationary phase.
section for 10 to 60 minutes. After washing, the slides Changing the chemical properties of the mobile phase
can be counterstained to visualize unstained structures. will selectively elute the trapped sample ions. Another
The stained sections are then dehydrated with 100% selective solid phase, affinity, is designed to immobi-
ethanol and air-dried. lize specific ions while other ions are washed through.
IHC has been a standard in pathology for many The selected ion is eluted by changing the mobile-phase
years.7 It provides the advantage of integrating target conditions.
detection, localization, and quantification in the context When a test sample is dissolved in the mobile phase,
of tissue morphology. Targeted therapeutic agents have it is introduced to HPLC by injection with a syringe
increased the use of IHC to assess tissue expression of through an injection port in the column. Pumps will
the target molecules. This guides treatment strategy at a force the sample through the column, and elutions are
relatively low cost compared with other methods. monitored by a detector that produces a readout of signal
peaks as the sample components elute. There are many
types of detectors, depending on the characteristics of
High-Performance Liquid
the sample, including light scattering, fluorescence,
Chromatography (HPLC)
refractive index, UV light absorption, and MS. The
Liquid chromatography was first designed to separate retention time and size of peaks indicate the type and
pigments extracted from plants using a solvent that amount of sample components. HPLC may be used to
was poured into a glass column packed with chalk separate nucleic acids as well as proteins.9
and alumina. The Russian botanist Mikhail S. Tswett Higher powered ultra-HPLC (UHPLC) columns have
named this process chromatography from the Greek been devised to increase resolution and lower separation
words chroma, meaning “color,” and graph, meaning time while using less solvent.10 UHPLC uses smaller-
“writing.”8 diameter columns and smaller stationary-phase parti-
HPLC is the basis for separation/analysis instruments cles than HPLC, with faster flow rates, up to 5 mL/min.
such as amino acid analyzers. Migration rates of mol- UHPLC is not recommended for complex, unfiltered
ecules differ with varying combinations of HPLC com- samples.
ponents and detectors. HPLC consists of two phases, a
mobile phase or solvent that flows through a stationary
Gas Chromatography
phase or solid support. The solvent chemistry is selected
so that it interacts more strongly with target molecules In GC, which is an automated method of analysis, the
to slow their migrations or more weakly to speed their mobile phase is an inert gas, and the stationary phase is
migrations. Organic solvent in the mobile phase may a high-boiling-point liquid that is absorbed to an inert
be the same throughout the column (isocratic) or of solid support in the column (Fig. 8.4). The sample is
increasing strength (gradient). The stationary phase will introduced to the column and vaporized into a gas. The
also interact with the sample and affect migration speed. inert gas carries the sample through the column. The
Molecules can thus be identified from normal migration strength of the interaction or dissolution of the sample
patterns. components into the liquid phase will result in varying
Absorbance
Time (minutes)
FIGURE 8.3 Liquid chromatography is Detector
the separation of molecules in solution
through interaction with a solid support in
the column. Bead or particulate matrices
have small spaces that hold and impede
movement of small particles. Larger parti-
cles migrate around the beads. Separated
molecules are detected directly or collected
fractions are further analyzed by MS.
Injection Mass Spectrometry

port
A mass spectrometer converts molecules to ions that can
be moved in a magnetic field based on their charge and
mass. In this automated method, an ion source sends
high-energy electrons that hit the target sample mol-
Carrier ecules, separating them into ions, usually cations with
Detector
gas the loss of one or two electrons. The ions themselves
may further fragment into smaller ions and neutral
FIGURE 8.4 Gas chromatography (gas-liquid chromatog- particles. This collection of particles is accelerated
raphy) is the separation of vaporized sample through a column and focused into a beam through a magnetic field that
of inert carrier gas and liquid stationary phase that differen- deflects the ions according to their mass and charge. By
tially adsorbs molecules. The sample is injected through the varying the strength of the magnetic field, the ions are
injection port, and the separated molecules are recorded by the aimed at a detector. The readout of the instrument is a
detector.
spectrum with the mass/charge value on the x-axis and
the abundance of the ion on the y-axis (Fig. 8.5). Mol-
ecules can therefore be identified by their characteristic
retention times in the column. The effluent from the spectrum or set of peaks.
column is monitored by a detector, such as a flame ion- Two ionization methods have been developed for
ization detector, that is sensitive to organic molecules. MS of large biomolecules such as proteins: electrospray
Alternatively, GC may be coupled to a mass spectrom- ionization (ESI) and matrix-assisted laser desorption/
eter. GC is used for detection of drugs and poisons and ionization (MALDI). Both result in ion formation with
their metabolites in biological samples.11,12 Coupled with limited loss of sample integrity. In ESI, a test sample is
MS, GC is being used to detect biomarkers of disease.13,14 converted into a fine spray of charged droplets that are
Sample
plate
Laser
A ⫹ 15 H⫹ AH
Relative intensity
A2H⫹2
Relative amount
A3H⫹3
A ⫹ 16 H⫹ Ionized
sample
A ⫹ 14 H⫹
molecules
m/z
Sample
A ⫹ 13 H⫹ FIGURE 8.6 In MALDI spectrophotometry, sample is

adhered to the matrix on a sample plate, ionized (protonated),
and released by laser bombardment. The ionized molecules are
separated in an electric field by mass and charge. A sample
molecule (A) will have multiple protonated species (AH, A2H,
m/z A3H).
FIGURE 8.5 In ESI spectrophotometry, multiple ionized

Ion
species from one protein are separated by mass and charge. source Detector
The results are plotted as mass/charge ratio (m/z) and relative
abundance.
electrostatically directed to the mass spectrometer inlet

through a vacuum where ions are released and drawn
through electrostatic lenses to the MS inlet. MALDI15
produces ions by firing a laser pulse into the sample
coated with a matrix (organic compound). Desorption
produces a positive ion from the sample molecule (Fig. FIGURE 8.7 In MALDI-TOF spectrophotometry, the ionized
8.6). The ion flies to the detector at a speed (time of molecules are accelerated at a fixed point and allowed to drift
through the flight tube to the detector. The time of flight is
flight) based on its mass and charge. High-molecu-
determined by the mass/charge ratio of the molecules. DNA
lar-weight molecules are identified using time of flight
sequence variants are detected as extension products of differ-
with MALDI ionization (MALDI-TOF). If the ions are ent sizes (mass).
allowed to drift in the flight tube, they will separate
according to mass/charge ratio so that the lighter ions
travel faster and reach the detector before the heavier
ions do (Fig. 8.7). An extension of MALDI, surface-en- compound is gained by three-dimensional quad-
hanced laser desorption/ionization (SELDI), combined rupolar electric fields, tandem MS (MS/MS), and
with time of flight (SELDI-TOF) offers flexibility in the hybrid instruments such as MALDI-TOF/TOF.
identification and quantification of peptides. The resolution of these instruments is sufficient to
identify amino acid sequences in peptides.
Advanced Concepts
Nucleic acids can also be analyzed by MS. Single-
It is possible to analyze almost any class of base-pair changes can be detected using a primer exten-
molecule with mass spectrometry techniques. sion technique. In this method, primers are bound to
Further information about the basic structure of a test DNA template just adjacent (5′) to the base posi-
tion to be analyzed. Extension of the primer with a
dideoxynucleotide (ddNTP) terminates the extension at especially for high-throughput procedures. A number of
one base and changes the mass and charge of the primer. techniques have been designed for the detection of DNA
The primer extension will only occur if the incoming mutations, from single-base-pair changes to large chro-
ddNTP is complementary to the template base next to mosomal rearrangements, without having to determine
where the primer is bound. By MALDI-TOF, the mass the primary DNA sequence. Some of these methods are
of the extended primer indicates the sequence or variant described in the following sections.
at the template site. Software automatically translates Sequence detection methods can be generally classi-
the mass into a genotype. Multiple variants can be ana- fied according to three broad approaches: hybridization-
lyzed simultaneously in a 384-well plate format. based methods, sequence (polymerization)-based methods,
and enzymatic or chemical cleavage methods. Brief de-
scriptions of representative methods are presented in the
Nucleic Acid Analyses
following sections. The methods selected are currently
Biochemical detection and/or characterization of pro- used or proposed for use in clinical applications. A sum-
teins using the biochemical methods just described may mary of the methods discussed in this chapter is shown
be part of a clinical chemistry laboratory menu or per- in Table 8.2.
formed as molecular diagnostics. Mutation detection
by analysis of nucleic acids is considered the classical
Hybridization-Based Methods
molecular methodology.
Nucleic acid analysis is performed on a variety of Single-Strand Conformation Polymorphism (SSCP)
specimen types. Inherited mutations are detected from SSCP is based on the preference of DNA (as well as
the most convenient and noninvasive specimen material, RNA) to exist in a double-stranded, rather than single-
such as blood or buccal cells. Somatic mutations are stranded, state.16,17 In the absence of a complementary
often more challenging to find because cells harboring strand, nucleic acids form intrastrand duplexes to attain
mutations may be only a small fraction of the total spec- as much of a double-stranded condition as possible.
imen that consists of mostly normal cells. Polymerase Each folded strand forms a three-dimensional structure,
chain reaction (PCR) amplification, which is part of or conformer, the shape of which is determined by the
many procedures, has simplified mutation detection, primary sequence of the folded strand. The migration of
especially from limiting specimens. PCR or other ampli- the single-stranded conformers in polyacrylamide gels
fication methods, however, must be performed under under precisely controlled denaturing and temperature
conditions that minimize the introduction of mutations conditions distinguishes sequence variants.
in the course of amplification. Although advances in For SSCP, dilute concentrations of short, dou-
genomic analysis have lowered the cost and increased ble-stranded PCR products, optimally 100 to 400 base
the resolution and throughput of mutation detection, pairs (bp) long, are denatured (e.g., in 10 to 20 mM
methods targeting single variants remain a rapid and NaOH, 80% formamide for 5 minutes at 95°C; or
cost-effective approach. 10 to 20 mM NaOH, 0.004 mM EDTA, 10% formamide
Interpretation of the results of such targeted mutation for 5 minutes at 55°C to 60°C), followed by rapid cooling.
analyses may also be challenging. Mutation scanning Because the diluted single strands cannot easily find their
by methods that do not indicate the primary sequence homologous partners, they fold by intrastrand hybridiza-
change do not differentiate between silent, conservative, tion, forming three-dimensional conformers. The shape of
and nonconservative mutations. The actual effect on the conformer depends on the complementary nucleotides
phenotype is left to posttest interpretation of supporting available for hydrogen bonding and folding. A single-base
clinical data and patient family history. Mutations dis- difference in the DNA sequence can cause the conformer
covered through this type of scanning may be subjected to fold differently. These conformers are resolved in a
to sequence analysis to confirm and further characterize polyacrylamide gel or by capillary electrophoresis
the mutated region. with strict temperature control. The speed of migration
Although it is the most definitive method for detect- depends on the shape as well as the size of the conformer.
ing mutations, DNA sequencing may not be appropriate, Differences in the shape of the conformers (kinks, loops,
TABLE 8.2 Summary of Mutation Detection Methodologies
Method* Target† (bp) Accuracy‡ (%) Specificity§ (%) Sensitivity|| (%)
Sequencing >1000 100 100 10–20
SSCP 50–400 70–100 80–100 5–20
ASO Defined 100 90–100 5–20
HR-MCA Defined 95–100 95–100 1–5
DHPLC 50–1000 95–100 85–100 5–20
Array technology Defined, multiplex 95–100 80–100 1–5
SSP Defined 98–100 95–100 0.0005
Allelic discrimination Defined 95–100 90–100 0.0001
PCR-RFLP Defined 100 100 0.01–1
Cleavase Defined, multiplex 100 95–100
*See text. Data are from methods done under optimal conditions.
†
Optimal length of sequence that can be screened accurately; defined methods target a single nucleotide or site; multiplex methods target multiple defined
types in the same reaction.
‡
Concordance with direct sequencing or other assays reported in the references.
§
True positive detection of mutations without concurrent false positive.
||
Detection of one mutant target in a background of normal targets.
Normal DNA Mutated DNA

Normal/
Normal Mutant mutant
Denaturation
and dilution Normal
A
Mutant
Gel electrophoresis Normal/

mutant
Electrophoresis Capillary electrophoresis
C
B
FIGURE 8.8 Single-strand conformation polymorphism analysis. Double-stranded PCR products (A) of normal or mutant
sequences are denatured and form conformers (B) through intrastrand hydrogen bonding. These conformers can be resolved (C) by
gel (left) or capillary (right) electrophoresis.
bubbles, and tails) are caused by sequence differences As a consequence, less sensitive stains such as ethidium
in the DNA single-strand nucleotide sequence (Fig. 8.8). bromide are not often used for this assay. Band or peak
The band or peak patterns are detected by silver stain, patterns different from those of normal sequence-control
radioactivity, or fluorescence. The use of low concen- conformers prepared simultaneously with the test con-
trations avoids the renaturation of homologous partners. formers indicate the presence of gene mutations.
SSCP is reported to detect 35% to 100% of putative in a 96-well plate format, similar to the capture probe
mutations.18 The assay can be sensitive enough to detect methods developed for detection of Chlamydia tracho-
mutations in samples containing as low as 5% poten- matis and Mycobacterium tuberculosis. For mutation
tially mutant cells,19 although specimens that are at least analysis, mutant or normal probes were immobilized on
30% potentially mutant cells produce more reliable a membrane. The sequence to be tested was amplified by
results. This requirement is satisfied in inherited muta- PCR with one regular and one biotinylated primer. The
tions because at least 50% of cells of a specimen will denatured biotinylated products were then exposed to
potentially carry a mutation. However, for somatic muta- the immobilized probes under conditions set so that only
tions, such as the analysis of tumor cells, the potentially the exact complementary sequences would hybridize.
mutant cells may be mixed with or surrounded by a vast Bound probes were detected with a conjugated horse-
majority of normal cells or tissue. Therefore, a cell sus- radish peroxidase-antibiotin Fab fragment and exposure
pension that is at least 30% tumor cells or a microdissec- to a chromogenic or chemiluminescent substrate. Gener-
tion of solid tumor tissue from fixed or frozen sections ation of a color or light signal indicated the binding of
is recommended. the test DNA to the normal or mutant probe. ASO has
been applied to human leukocyte antigen (HLA) typing,
but it has now been replaced with microarray and bead
Advanced Concepts array methods and, more recently, by next-generation
sequencing.25
Because SSCP worked more accurately on some
genes than others, modifications of the SSCP Melt-Curve Analysis
procedure were developed, for instance, using Like ASO, melt-curve analysis (MCA) exploits the
RNA instead of DNA (RNA-SSCP or rSSCP)20,21 sequence- and stacking-directed denaturation character-
or using restriction endonuclease fingerprint- istics of DNA duplexes.26 The method is a postamplifica-
ing (REF-SSCP).22 The rSSCP and REF-SSCP tion step of real-time PCR. PCR amplicons generated in
methods, although more sensitive, were more dif- the presence of a DNA-specific fluorescent dye, such as
ficult to interpret and were not put into general ethidium bromide, SYBR green, or LC green, are heated
use. at a rate of about 0.3°C/sec. The dyes, specific for dou-
ble-stranded DNA, initially yield a high signal because
the DNA is mostly double stranded at the low tempera-
ture (50°C to 60°C). As the temperature rises, the DNA
Allele-Specific Oligomer Hybridization
duplexes begin to separate into single strands, losing
Allele-specific hybridization, or allele-specific oligo-
dye accordingly. The fluorescent signal gives a pattern
mer hybridization (ASO), utilizes the differences in
as shown in Figure 8.9. Sequence differences result in
the melting temperatures of short sequences of about
20 bases with one or two mismatches and those with
Homozygous
no mismatches. Synthetic single-stranded probes with %DS
normal
the normal or mutant target DNA sequence are used for
this assay. At specific annealing temperatures and condi- Heterozygous
tions (stringency), a probe will not bind to a near com- DS=SS
plementary target sequence with one or two mismatched Homozygous
bases, whereas a probe with a perfect complementary mutant
sequence will bind. ASO uses an immobilized target and %SS
a labeled probe in solution. This dot blot method was 50 60 70 80
used to test for known, frequently occurring mutations, Temperature (°C)
for example, in the BRCA1 and BRCA2 gene mutations FIGURE 8.9 MCA of homozygous mutant, heterozygous,
frequently observed in inherited breast cancer23 and and normal PCR products. As the temperature increases, dou-
the p16 gene mutations in familial melanoma.24 ASO ble-stranded (DS) DNA denatures into the single-stranded (SS)
analysis also may be carried out as a reverse dot blot state.
Probes
Normal
Heterozygous
mutation
df/dt
Tm = 62°C
Target sequence DNA
Temperature
FIGURE 8.10 A plot of the derivative of the fluorescence Tm = 55°C

data (df/dt) versus temperature shows the inflection point of Mutation
the melt curve as a peak at the Tm of the test sequence. A Normal
Heterozygous
normal homozygous sample should have a Tm that can be dis-
Fluorescence
mutant
tinguished from that of the mutant sequence.
Homozygous
mutant
different melting characteristics and melting tempera-

tures (Tms; where there are equal amounts of double-
and single-stranded DNA) for each sequence. The Tm
Fluorescence
is illustrated as a peak, plotting the derivative (speed of
(d/dT)
decrease) of fluorescence versus temperature. The results
are interpreted by the temperature peak placement with
respect to the temperature on the x-axis. Specimens with 55° 62°
identical sequences should yield overlaying peaks at the Temperature
expected Tm, whereas specimens containing different A FRET probes
sequences will yield two or more peaks at different tem-
peratures (Fig. 8.10). FIGURE 8.11 MCA with FRET probes. A mismatch between
The nonspecific dyes used in MCA are not sequence the target and probe will lower the Tm of the duplex.
specific and therefore do not distinguish between the
target amplicon and extraneous products that may occur
in the PCR reaction, such as primer dimers or mis- and the probe, hydrogen bonding is perturbed between
primed amplicons. Although the target sample should the two strands of the double helix. The mismatch
be identifiable by its Tm, such unintended products can decreases the dissociation temperature compared with
complicate the melt curve and confuse interpretation. matched or complementary sequences. A Tm lower than
The specificity of MCA is increased by using high- that of the probe and its perfect complement, therefore,
resolution melt-curve analysis (HR-MCA),27,28 which indicates the presence of a mutation, or sequence differ-
uses fluorescent resonance energy transfer (FRET) ence, between the probe reference sequence and the test
probes that hybridize next to one another across the sequence.
sequence position being analyzed. The probes fluoresce
only when bound to the target sequence because FRET
fluorescence relies on the transfer of energy from a donor Advanced Concepts
fluorescent molecule (fluor) on one probe to an acceptor
fluor on the other probe. As the temperature increases, PCR products smaller than 300 bp in size are pre-
the probes dissociate at a specific Tm. When the probes ferred for MCA. The ability of the assay to dis-
dissociate from the target, the donor is no longer close tinguish sequence differences decreases with the
to the acceptor, and the fluorescence drops (Fig. 8.11). If increasing size of the PCR product.27
the target sequence has a mismatch between the target
Probe Special instrumentation is required for MCA and

HR-MCA. Thermal cyclers with fluorescent detection
have melt-curve options that can be added to the thermal
cycling program. Instruments that perform MCA only
can process more samples per unit time than the thermal
cycler systems. Melt-curve methodology has been pro-
posed for a variety of clinical laboratory applications,
such as detection of DNA polymorphisms and typing of
microorganisms. Naturally occurring sequence variation
must be considered in performing MCA because it can
complicate the interpretation of the test.
Heteroduplex Analysis
Solution hybridization and electrophoresis of test
nucleic acid fragments mixed with reference nucleic
acid fragments can reveal mutations. To form hetero-
duplexes, nonidentical double-stranded DNA duplexes
are heated to a temperature that results in complete
denaturation of the double-stranded DNA (e.g., 95°C)
and then slowly cooled (e.g., −1°C/4 to 20 sec). Het-
eroduplexes are formed when single strands that are not
completely complementary hybridize to one another
(Fig. 8.13). (Heteroduplexes are also formed when test
Temperature PCR products amplified from genetically heterozygous
B Simple probe
specimens are denatured and renatured.) The hetero-
duplexes migrate differently than do homoduplexes
FIGURE 8.12 MCA with a single probe. The binding of the through polyacrylamide or agarose gels. The presence of
single probe and fluorescence will decrease with increasing bands different from a homozygous reference control is
temperature. The rate of decrease will depend on the hybrid- indicative of mutations. Gel-based heteroduplex methods
ized sequences.
have been designed for HIV typing and hematological
testing.
Heteroduplexes are also resolved by denaturing high-
FRET is most frequently performed with two probes; performance liquid chromatography (DHPLC). This
however, single-probe systems have been developed. version of heteroduplex analysis is performed on
The single probe is designed to fluoresce much more PCR products, ideally 150 to 450 bp in length. HPLC
brightly when hybridized to the target. The fluorescence separation is performed on a 25% to 65% gradient of
is lost on dissociation (Fig. 8.12). Another modification acetonitrile in triethylammonium acetate at the melting
that is reported to improve the sensitivity of MCA is the temperature of the PCR product. The heteroduplexes
covalent attachment of a minor groove binder (MGB) elute ahead of the homoduplexes as the denaturing con-
group to the probe. The MGB, dihydrocyclopyrroloin- ditions intensify. The migrating homoduplexes and het-
dole tripeptide, folds into the minor groove of the duplex eroduplexes are detected by absorbance at 260 nm or
formed by hybridization of the terminal 5 to 6 bp of the by fluorescence. HPLC methods were reported to be
probe with the template. This raises the melting tempera- more sensitive than gel methods, with greater capacity
ture of the probe, especially one with high A/T content. for screening large numbers of samples. Although HPLC
The Tm of a 12- to 18-bp MGB conjugated probe was analysis of heteroduplexes was evaluated as a mutation
measured to have hybridization properties equivalent to screening method, gel-based heteroduplex analyses are
that of a 25- to 27-bp non-MGB probe.29 still routinely used in the clinical laboratory.
T Array Technology
A
Single-base-pair resolution by hybridization differences
is achievable with high-density oligonucleotide arrays.
C
G These methods are similar to comparative genome
Heterozygous sample hybridization but focus on single genes with higher res-
or sample + probe olution, as in ASO procedures. The advantage of array
methods is the large number of inquiries (potential
Denature sequence mutations or SNPs) that can be tested simul-
taneously. For analysis, the test DNA is fragmented by
T
treatment with DNase before binding to the complemen-
A tary probes on the array. If the sample fragments are too
C large (not treated with DNase), a single-base-pair mis-
G
match has minimal effect on hybridization because the
fragment binds to multiple probes and the specificity of
Renature detection is lost. An example of one type of hybridiza-
T
T tion format, standard tiling, is shown in Figure 8.14.
A
G
In this format, the base substitution in the immobilized
probe is always in the 12th position from its 3′ end. Com-
C
C monly occurring mutations are targeted in another type
G
A of format, redundant tiling, in which the same mutation
Homoduplexes Heteroduplexes is placed at different positions in the probe (at the 5′ end,
FIGURE 8.13 Heteroduplex analysis is performed by mixing in the middle, or at the 3′ end). After hybridization of the
sample amplicons with a reference amplicon, denaturing, and fluorescently labeled sample DNA, the fluorescent signal
slowly renaturing. If the sample contains mutant sequences, a is read on a scanner with appropriate software, and the
fraction of the renatured products will be heteroduplexes. mutations are identified as indicated by which probes are
These structures can be resolved from homoduplexes by bound. Although not performed routinely in clinical lab-
electrophoresis. oratories, a number of applied methods were developed
C A T C G/A T
A C G T Del A C G T Del A C G T Del A C G T Del A C G T Del A C G T Del
Normal Heterozygous mutation

FIGURE 8.14 Mutation analysis of the p53 gene by high-density oligonucleotide array analysis. Each sequence position is rep-
resented by 10 spots on the array, 5 sense and 5 antisense probes. The sequence binds only to its perfect complement probe. The
illustration shows three adjacent sequence positions, CAT. Binding of the sample fragment is detected by increased fluorescence.
A fragment with the normal sequence is on the right; a heterozygous mutation is on the left.
Locus-specific sequences Primer Amplification

5′ G 3′
3′ C 5′
Normal template
Primer-binding site 3′ No amplification

Beads 5′ G
3′ T 5′
Mutant template
A FIGURE 8.16 Sequence-specific primer amplification. Suc-

G
cessful amplification will occur only if the 3′ end of the primer
matches the template.
Cy3– (A) sequences, the combination of bead color and test label
Or
reveals the presence or absence of a mutation or poly-
morphism. The advantage of this arrangement is that
Cy5– (G) multiple loci can be tested simultaneously from small
samples. Up to 100 analytes are tested in a single well of
a microtiter plate. This method requires a flow cytome-
Cy3– try instrument that excites and reads the emitted fluores-
A allele cence as the beads flow past a detector. This technology
Or
has been applied to antibody detection and infectious
Cy5– diseases and is used in tissue typing and in other clinical
G allele
applications.
FIGURE 8.15 Bead array technology. Beads colored with
distinct fluorescent dyes (upper left) are covalently attached to Sequencing (Polymerization)-Based Methods
the probe sequences, with each color of bead attached to a
probe representing a specific locus. In a sequence-specific Sequence-Specific (Primer) PCR
PCR, test DNA is amplified with tailed primers. The tailed Sequence-specific (primer) PCR (SSP-PCR) is com-
PCR products are amplified in a second reaction to generate monly used to detect point mutations and other SNPs.
labeled amplicons that will bind to specific beads, according to There are numerous modifications to the method, which
the gene locus. The combination of bead label and the hybrid- involve the careful design of primers such that the primer
ized amplicon label reveals whether there is a mutant or normal 3′ end falls on the nucleotide to be analyzed. Unlike the
allele at that locus. 5′ end, the 3′ end of a primer must match the template
perfectly to be extended by Taq polymerase (Fig. 8.16).
By designing primers to end on a mutation, the presence
using high-density oligonucleotide and microelectronic or absence of product is interpreted as the presence or
arrays.30,31 absence of the mutation.
Bead array technology utilizes sets of color-coded In a modification of SSP-PCR, normal and mutant
polystyrene beads in suspension as the solid matrix sequences are distinguished from one another by
(Fig. 8.15). In an extension of a flow cytometry system,32 increasing the length of the normal or the mutant
100 sets of beads are dyed with distinct fluorochrome primer, resulting in differently sized products, depend-
mixes. Each set is coated with oligonucleotide probes ing on the sequence of the template (Fig. 8.17). Alter-
corresponding to a genetic locus or gene region. In this natively, primers can be multiplexed (Fig. 8.18).
technology, 105 or more probes are attached to each 3- to Multiplexed SSP-PCR was originally called amplifi-
6-micron-diameter bead. When labeled test samples are cation refractory mutation system PCR or tetra-primer
hybridized to the beads through complementary probe PCR.33 Sequence-specific PCR is routinely used for
Primer Primer 2 (specific for

Primer 1 normal sequence)
3′ C 5′ G
5′ T 3′ 3′ C 5′
3′ A 5′
Primer specific Template Primer 3 (specific for mutations)
C for mutation (T) A
Primer specific for
normal sequence (C) 3′ G 5′
M Normal Heterozygous Homozygous Primer 4

mutant mutant
+ m m +
Mutant amplicon
Normal amplicon
FIGURE 8.17 Allele-specific primer amplification of a C→T

mutation. A longer primer is designed with the mutated nucle-
otide (A) at the 3′ end. This primer is longer and gives a larger
amplicon than the primer binding to the normal sequence (top).
The resulting products can be distinguished by their size on an 1–4
agarose gel (bottom). First lane: molecular-weight marker; 1–3
Specific for
second lane: a normal sample; third lane: a heterozygous mutation
2–4
mutant sample; fourth lane: a homozygous mutant.
high-resolution HLA typing and for detection of com-

monly occurring mutations.
A high-throughput application of bead array technol- FIGURE 8.18 Multiplex allele-specific PCR. The mutation
ogy34,35 uses sequence-specific PCR (Fig. 8.15). In this (C→A) is detected by an allele-specific primer (3) that ends at
the mutation. Primers 3 and 4 would then produce a mid-sized
assay, primers tailed at the 5′ end with locus-specific
fragment (1–3). If there is no mutation, a normal primer (2)
sequences and allele-specific sequences (A or G in
binds and produces a smaller fragment (2–4). Primers 1 and 4
Fig. 8.15) are used to amplify the test DNA. Each result- always amplify the entire region (1–4).
ing PCR product will have an allele-specific sequence
at one end and a locus-specific sequence at the other
end. The PCR products are subsequently amplified in a Allelic Discrimination With Fluorogenic Probes
second round using Cy3 or Cy5 (fluorescently) labeled Thermal cyclers with fluorescent detection support
5′ primers, complementary to the sequences introduced allelic discrimination with fluorogenic probes. This
by the tailed primers in the first round. These ampli- method is a real-time PCR assay, using two probes
cons are then hybridized to the locus-specific sequences labeled 3′ quencher molecules and different fluors on
covalently attached to the bead array. The bead color the 5′ ends (Fig. 8.18). Each probe is complementary
(locus) combined with Cy3 or Cy5 fluorescence (allele) to either the normal or mutant sequence. The hybridized
types the allele at each locus. This system is one of the probe is digested by the polymerase enzyme, releasing
technologies that was used in the Human Haplotype the reporter dye. The presence of the corresponding flu-
Mapping Project. orescent signal indicates whether the test sequence is
Normal probe (FAM) Mutant probe (VIC)
Taq Taq
Normal
allele
(FAM)
Mutant
allele
(VIC)
FIGURE 8.19 Allelic discrimination using fluoro-

genic probes. Probes, complementary to either the
Mutant allele (VIC)

normal sequence (left) or the mutant sequence
(right), are labeled with different fluors, for example,
FAM and VIC, respectively. The Taq exonuclease
functions only if the probe is matched to the
sequence being tested. High FAM indicates a normal
sequence, and high VIC indicates a mutant sequence.
If both fluors are detected, the test sample is
heterozygous. Normal allele (FAM)
normal or mutant, that is, whether the probe matched Normal …GTCAGGGTCCCTGC…
and hybridized to the test sequence. In the example
Mutation
shown in Figure 8.19, the probe complementary to the …GTCAGGATCCCTGC…
normal sequence is labeled with FAM dye; the probe

complementary to the mutant sequence is labeled with Normal Mutant Het
VIC dye. If the test sequence is normal, FAM fluores- U B U B U B
cence will be high, and VIC fluorescence will be low. If
the test sequence is mutant, VIC will be high, and FAM
will be low. If the sequence is heterozygous, both VIC
and FAM will be high. Negative controls show no VIC
or no FAM. This assay has the advantage of interrogat-
ing multiple samples simultaneously and was proposed
as a practical high-throughput laboratory method.36,37
Enzymatic and Chemical Cleavage Methods FIGURE 8.20 PCR-RFLP. The normal sequence (top line) is
converted to a BamH1 restriction site (GGATCC) by a G>A
Restriction Fragment Length Polymorphisms mutation. The presence of the mutation is detected by testing
If a mutation changes the structure of a restriction the PCR product with BamH1. The bottom panel shows the
enzyme target site or changes the size of a fragment predicted gel patterns for the homozygous normal, homozy-
generated by a restriction enzyme, restriction fragment gous mutant, and heterozygous samples uncut (U) or cut with
length polymorphism (RFLP) analysis can be used to BamH1 (B).
detect the sequence alteration. To perform PCR-RFLP,
the region surrounding the mutation is amplified, and the PCR product results in cutting of the mutant ampli-
mutation is detected by cutting the amplicon with the con, but not a normal control amplicon or vice versa.
appropriate restriction enzyme (Fig. 8.20). Mutations Although straightforward, PCR-RFLP requires careful
may inactivate a naturally occurring restriction site or design, because rare polymorphisms have been reported
generate a new restriction site so that digestion of the to confound RFLP results. Several PCR-RFLP methods
+ + + m the amplicons. The PCR reaction and the HindIII diges-

+ m + m tion are performed in the same tube, and the products
are separated on one lane of the gel.39 This procedure
is used in the clinical analysis of factor V Leiden and
prothrombin mutations.
Prothrombin
Nonisotopic RNase Cleavage Assay
Nonisotopic RNase cleavage assay (NIRCA) is a het-
Factor V eroduplex analysis using duplex RNA.40,41 The sequences
to be scanned are amplified using primers tailed with
promoter sequences of 20 to 25 bp. T7 or SP6 phage
RNA polymerase promoters are most often used for this
+ + m + purpose. Following amplification, the PCR products
+ m m + with the promoter sequences are used as templates for in
vitro synthesis of RNA with the T7 or SP6 RNA poly-
merase enzymes. This reaction yields a large amount
Prothrombin of double-stranded RNA (Fig. 8.22). The transcripts
are denatured at 95°C and then renatured by cooling
to room temperature. If a mutation is present, hetero-
duplexes form between normal and mutant transcripts.
1-bp mismatch
Factor V These mismatches in the RNA are targets for cleavage
by RNase enzymes. A mixture of single-strand-specific
E. coli RNase I and Aspergillus RNase T1 cleaves differ-
ent types of mismatches. The remaining double-stranded
3-bp mismatch
RNA fragments can then be separated by agarose gel
FIGURE 8.21 Multiplex PCR with mutagenic primers to electrophoresis. As in DNA heteroduplex analysis, the
detect mutations in factor V and prothrombin. The primer size of the RNA fragments indicates the placement of
sequences are designed to generate a HindIII site in the PCR the mutation. NIRCA has been applied to screening of
product if the mutations are present. The prothrombin and
several clinical targets, including factor IX,7 TP53, Jak2,
factor V PCR products are different sizes that can be resolved
on the gel in a single lane.
and BRCA1.
Cleavase Assay
The Cleavase assay is based on the characteristic enzy-
matic activity of a proprietary enzyme system (Cleav-
are widely used for detection of commonly occur- ase).42,43 Premixed reagents including Cleavase are added
ring mutations, such as FLT3 kinase domain and HFE to a standard 96-well plate along with the test specimens
mutations. (sample DNA or PCR products) and controls. Cleavase
PCR-RFLP can be multiplexed to detect more than recognizes the structure formed by hybridization of the
one gene mutation simultaneously. This has been prac- normal or mutant probes to the test sequences. During
tical for the detection of separate gene mutations that an isothermal incubation, if the probe and test sequence
affect the same phenotype, for example, factor V Leiden are complementary, two enzymatic cleavage reac-
and prothrombin.38 Alternatively, a combination of tions occur, ultimately resulting in a fluorescent signal
SSP-PCR and PCR-RFLP is also applied to simultane- (Fig. 8.23). The signal can be read by a standard flu-
ous detection of mutations in more than one locus. An orometer. The advantages of this method are the short
example is shown in Figure 8.21, in which a primer hands-on time and optional PCR amplification. This
designed to produce a restriction site in the amplicon is method has been applied to several areas of clinical
used for each gene in a multiplex PCR. In the example, molecular diagnostics, including genetics, hemostasis,
the primers are designed to generate a HindIII site in and infectious disease.
Normal Mutant
Tailed primer
PCR
RNA polymerase
PCR products
Transcription
RNA that
hybridizes to
make double
strands
Denaturation,
reannealing
Single strand-specific RNase
FIGURE 8.22 NIRCA analysis. Normal (left)

and mutant (right) transcription templates cov-
ering the area to be screened are produced by
Normal Mutant
PCR with tailed primers carrying promoter
sequences. RNA polymerase then transcribes
the PCR products. The transcripts are dena-
tured and reannealed, forming heteroduplexes Normal
transcript
between normal and mutant transcripts. RNase Cleaved
cleavage products can be resolved on native mutant
agarose gels. transcript
modified primers. As the field of molecular diagnostics

Other Methods
grows, the development of high-throughput methods has
The challenges of clinical laboratory requirements for become a main focus. Array-based methods and massive
robust, accurate, and sensitive assays have driven the parallel sequencing methods now provide the specific
discovery of new techniques and modification of exist- multiplex detection and sensitivity required for clinical
ing techniques. As a consequence, many methods have applications. Instrument and reagent costs, which were
been devised, especially for high-throughput screening. relatively high for these technologies, are decreasing,
SSCP was a commonly used mutation screening method especially relative to the information generated per test.
in clinical laboratories, but it became apparent from the Overall, the method selected will depend on the avail-
use of SSCP that a single procedure may not be ideal for able instrumentation, the genetic target, and the nature
all genes—hence the development of diverse methods. of the mutation.
Combinations of methods have also been proposed to A summary of the methods described in this chapter
increase sensitivity and detection, such as RFLP with is outlined in Table 8.2. The performance of each method
Flap Cleavage Flap No cleavage

Mutant Mutant
Invader A Invader
probe G probe
probe probe
T T
Mutant
Cleavage test
F
DNA
A Q
Detection F
FIGURE 8.23 Cleavase single-color assay. Hybridization of supplied probe and anchor sequences to the input template (upper
left) forms a structure that is the substrate for the cleavage enzyme. The enzyme removes the flap sequences, which form another
hybridization structure with the labeled probe. The second cleavage reaction releases the fluorescent dye from the vicinity of the
quencher on the probe, a fluorescent signal. If the template does not match the probe in the first hybridization (upper right), no
cleavage occurs.
varies, depending on the specimen, template sequence, For DNA and cDNA, the first nucleotide of the
and type of mutation to be detected. For instance, first amino acid in the sequence, usually A of ATG for
methods that detect only mutations involving specific methionine, is designated as position +1. The preceding
nucleotides can have 100% accuracy and specificity for nucleotide is position -1. There is no nucleotide position
these mutations but 0% for mutations affecting other 0. Nucleotide changes are expressed as the position or
nucleotides. Procedures that are developed by targeting nucleotide interval, the type of nucleotide change, the
specific mutations will perform for that target gene or changed nucleotide, the symbol >, and finally the new
region but may not work as well for other targets. For nucleotide. For example, consider a nucleotide reference
instance, hybridization methods may detect mutations sequence: ATGCGTCACGAATTA. A substitution of a
in GC-rich sequence environments more accurately than T for a C at position 7 in the DNA sequence (mutant
in AT-rich sequences. Single-target methods are useful sequence ATGCGTTACGAATTA) is expressed as
low-cost screening methods, but comprehensive and c.7C>T; the “c.” is for coding sequence. This format is
definitive methods such as direct sequencing are cur- intended to distinguish nucleotide changes from amino
rently preferred, especially with complex diseases and acid changes in proteins and is recommended for test
new treatment strategies revealing growing numbers of reports. In large variant databases, such as those used for
clinically important variants. NGS analysis, however, the format c.C7T may be used.
A deletion of nucleotides 6 and 7, ATGCG__
ACGAATTA, is expressed as c.6_7del or c.6_7delTC.
GENE VARIANT NOMENCLATURE An insertion of a TA between nucleotides 5 and 6, ATG-
CGTATCACGAATTA, is denoted c.5_6insTA. Dupli-
Accurate testing and reporting of gene mutations require cations are a special type of insertion. For example, a
a descriptive and consistent system of expressing muta- duplication of nucleotides 4 and 5, ATGCGCGTCAC-
tions and polymorphisms. Recommendations have been GAATTA, is expressed as c.4_5dupCG. An insertion
reported and generally accepted.44 This section fea- with a concomitant deletion, indel, has three alternate
tures general descriptive terms for basic alterations and descriptive terms. For example, if TC at positions 6 and 7
structures. of ATGCGTCACGAATTA is deleted from the reference
sequence and GACA is inserted, the altered sequence, and the inserted amino acids. For instance, insertion of
ATGCGGACAACGAATTA, is denoted as c.6_7delT- amino acids glycine (G), alanine (A), and threonine (T),
CinsGACA, c.6_7delinsGACA or c.6_7>GACA. Inver- making the altered amino acid sequence MRGATHEL,
sion of nucleotides is designated by the nucleotides is indicated by p.R2_H3insGAT or, alternatively, p.R2_
affected, “inv,” and the number of nucleotides inverted. H3ins3. A short notation for frameshift mutations is the
For example, inversion of GCGTCAC starting at posi- amino acid, its position, and “fs.” A frameshift muta-
tion 3 to position 9 in the reference sequence (ATCACT- tion affecting the histidine residue changing the amino
GCGGAATTA) is c.3_9inv7. acid sequence to MRCPLRGWX is simply p.H3fs, or
Gene mutations in recessive diseases, where both p.H3fs* because frameshift mutations result in ter-
alleles are affected, are indicated by the designation of mination within a few amino acids. The length of the
each mutation separated by +. Thus, a 2357C>T muta- shifted open reading frame is indicated by adding X
tion in one allele of a gene and a 2378delA mutation and the position of the termination codon. p.H3CfsX7
in the other allele on the homologous chromosome is is a frameshift in codon 3 that changes a histidine to a
written as c. [2357C>T]; [2378CdelA]. This is distinct cysteine and new reading frame ending in a stop at the
from two mutations in the same allele, which is written seventh codon.
as c.[2357C>T(;) 2378CdelA]. In cases of loss of het- To distinguish between mutation nomenclature refer-
erozygosity, [0] indicates the absence of the entire ref- ring to genomic DNA, coding (complementary or copy)
erence coding sequence on the other chromosome. For DNA, mitochondrial DNA, RNA, or protein sequences,
example, c.[2357G>A];[0] denotes a G to A change at the prefixes of “g.,” “c.,” “m.,” “r.,” and “p.” are rec-
nucleotide 2357 on one chromosome and the loss of the ommended, respectively. Furthermore, RNA sequences
gene on the homologous chromosome. are written in lowercase letters. For example, c.89T>C
Mutations in introns of genomic DNA are indicated in the coding DNA would be r.89u>c in RNA.
by the position of the mutation in the genomic sequence Complex changes and multiple concurrent mutations
of the DNA or the position from the end of the coding are reported as they occur. Some mutations, even with
sequence + the position in the intron. Thus, a G>T sequence information, cannot be positively determined
mutation 5 nucleotides into intron 2 that starts after the and must be inferred, for example, additions or deletions
91st nucleotide of exon 2 is designated as c.91+5G>T. of repeat units in repeated sequences. For these changes,
If this same base change was at the 356th nucleotide in it is assumed that the 3′-most repeat is the one affected,
the genomic sequence, an alternative designation would and the alteration is noted for that position. Updates
be g.356G>C for genomic sequence. and further clarifications of mutation nomenclature are
At the protein level, numbering begins with the initial continually being addressed. Current information and
amino acid, methionine, in the protein sequence desig- descriptors for more complex changes are available from
nated +1. The single-letter code has been used to convey the Human Genome Variation Society (HGVS) at http://
protein sequence, but because of potential confusion www.HGVS.org/varnomen.
with the single-letter designations, three-letter denota-
tions are also acceptable. Stop codons are designated by
X in either case. Amino acid changes are described by GENE NAMES
the amino acid changed, the position, and the new amino
acid. Consider the protein sequence methionine–argi- Gene nomenclature is different from gene names or
nine–histidine–glutamic acid–leucine, or p.MRHEL. If sequence designation. The Human Genome Organi-
the second amino acid, arginine (R), was substituted by zation (HUGO) gene nomenclature committee has set
serine (S), the mutation of the new amino acid sequence, rules for reporting or publishing gene names (see http://
p.MSHEL, would be p.R2S. A nonsense mutation in www.hugo-international.org/). Gene names should be
codon 4, mutant sequence MRHX, would be written as capitalized and set in italics with no hyphens. Protein
p.E4X, E4ter or E4*. Deletion of the arginine and his- names are not italicized nor completely capitalized. For
tidine, M__EL, would be p.R2_H3del or p.R2_H3del2. example, the KRAS gene codes for the K-Ras protein,
Insertions are denoted by the amino acid interval, “ins,” and the TP53 gene codes for the protein p53. Thus, a
report will contain the official gene name and the appro- 9. A reference sequence, ATGCCCTCTGGC,
priately named change in the DNA sequence, if present. is mutated in malignant cells. The following
mutations in this sequence have been
described:
STUDY QUESTIONS ATGCGCTCTGGC
ATGCCCTC - -GC
ATAGCCTCTGGC
1. What characteristic of the genetic code facilitates
ATGTCTCCCGGC
identification of open reading frames in DNA
sequences? Express these mutations using the accepted gene
nomenclature (A = nucleotide position 1).
2. Compare and contrast EIA with western blots for
the detection of protein targets. 10. A reference peptide, MPSGCWR, is subject
to inherited alterations. The following peptide
3. On a size-exclusion column, large molecules sequences have been reported:
will elute_______________ (before/after) small
MPSTGCWR
molecules.
MPSGX
MPSGCWLVTGX
4. MALDI methods separate ions by
MPSGR
a. molecular volume. MPSGCWGCWR
b. mass.
Express these mutations using the accepted
c. charge.
nomenclature (M = amino acid position 1).
d. mass and charge.
5. What is a heteroduplex?
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Chapter 9
DNA Sequencing
Outline Objectives
DIRECT SEQUENCING 9.1 List the components and the molecular reactions
Manual Sequencing that occur in chain termination sequencing.
Chemical (Maxam–Gilbert) Sequencing 9.2 Discuss the advantages of dye primer and dye
Dideoxy Chain Termination (Sanger) Sequencing terminator sequencing.
Automated Fluorescent Sequencing 9.3 Derive a text DNA sequence from raw sequencing
Approaches to Automated Sanger Sequencing data.
The Sequencing Ladder 9.4 Describe examples of alternative sequencing
Electrophoresis methods, such as pyrosequencing and next-
Sequence Interpretation generation sequencing (NGS).
PYROSEQUENCING 9.5 Show different technical approaches to NGS
BISULFITE DNA SEQUENCING and the two approaches used most in clinical
RNA SEQUENCING applications.
NEXT-GENERATION SEQUENCING 9.6 Describe how NGS sequencing libraries are made.
Gene Panels 9.7 Distinguish primer and probe-based enrichment.
NGS Library Preparation 9.8 Define bioinformatics, and describe electronic
Targeted Libraries systems for the communication and application of
Sequencing Platforms sequence information.
Sequence Quality 9.9 Recount the events of the Human Genome
Filtering and Annotation Project.
BIOINFORMATICS 9.10 Explain how variant databases were developed
THE HUMAN GENOME PROJECT following completion of the Human Genome
Variant Associations With Phenotype Project.
The Human Haplotype Mapping Project
The 1000 Genomes Project
223
DNA sequence information (the order of nucleotides in

the DNA molecule) is used in the medical laboratory
for a variety of purposes, including detecting mutations,
typing microorganisms, identifying human haplotypes, DMS FA H H+S
and designating polymorphisms. Treatment strategies
including targeted therapies are now selected based on
the results of these techniques.1
G G C C
G A T C
DIRECT SEQUENCING G A T C
G G C C
The importance of knowing the order, or sequence, of A T
G C
nucleotides on the DNA chain was appreciated in the G C
earliest days of molecular analysis. Elegant genetic A T
experiments with microorganisms indirectly detected
FIGURE 9.1 Chemical sequencing proceeds in four separate
molecular changes at the nucleotide level using pheno-
reactions in which the labeled DNA fragment is selectively
typic characteristics, such as nutrient requirements. broken at specific nucleotides. DMS, Dimethylsulphate; FA,
Indirect methods of investigating nucleotide sequence formic acid; H, hydrazine; H + S, hydrazine + salt.
differences are still in use today. Without knowing the
nucleotide sequence of the targeted areas, the results
from many of these methods would be difficult to inter- TABLE 9.1 Specific Base Reactions
pret; in fact, some methods would not be useful at all. in Maxam–Gilbert Sequencing
Direct determination of the nucleotide sequence, or DNA
sequencing, is the most definitive molecular method to Chain Time
identify genetic lesions. Breaks (min at
At: Base Modifier Reaction 25°C)
Manual Sequencing G Dimethylsulphate Methylates G 4
Direct determination of the order, or sequence, of nucle- G + A Formic acid Protonates 5
otides in a DNA polymer is the most specific and direct purines
method for identifying genetic lesions (mutations) or T + C Hydrazine Splits 8
polymorphisms, especially when looking for changes pyrimidine rings
affecting only one or two nucleotides. Two types of
sequencing methods were concurrently developed in C Hydrazine + salt Splits only 8
C rings
the 1970s: Maxam–Gilbert sequencing2 and Sanger
sequencing.3
(Fig. 9.1). Upon addition of a strong reducing agent,
Chemical (Maxam–Gilbert) Sequencing such as 10% piperidine, the single-stranded DNA would
break at specific nucleotides (Table 9.1).
The Maxam–Gilbert chemical sequencing method was
developed by Allan M. Maxam and Walter Gilbert.
Maxam–Gilbert sequencing required a double- or single- Advanced Concepts
stranded version of the DNA region to be sequenced,
with one end radioactively labeled. To make a radioactive sequence template, (32P)-
For sequencing, the labeled fragment, or template, ATP is added to the 5′ end of a DNA fragment,
was aliquoted into four tubes. Each aliquot was treated using polynucleotide kinase, or the 3′ end, using
with a different chemical with or without high salt
Chapter 9 • DNA Sequencing 225
pyrimidine (C + T) lane were called based on whether

terminal transferase plus alkaline hydrolysis to they were also present in the G- or C-only lanes. In that
remove excess adenylic acid residues. Double- way, the sequence was read from the bottom (5′ end of
stranded fragments labeled only at one end are also the DNA molecule) to the top (3′ end of the molecule)
produced by using restriction enzymes to cleave a of the gel.
labeled fragment asymmetrically, and the cleaved Although Maxam–Gilbert sequencing was a rela-
products are isolated by gel electrophoresis. Alter- tively efficient way to determine short runs of sequence
natively, denatured single strands are labeled data, the method was not practical for high-throughput
separately, or a “sticky” end of a restriction site sequencing of long fragments. In addition, the hazardous
is filled in, incorporating radioactive nucleotides chemicals hydrazine and piperidine required more elab-
with DNA polymerase. orate precautions for use and storage. The method was
therefore replaced by the dideoxy chain termination
sequencing method for most sequencing applications.
After the reactions, the fragments were separated by
size on a denaturing polyacrylamide gel. An example
of Maxam–Gilbert sequencing results is shown in Advanced Concepts
Figure 9.2. The sequence was inferred from the bands
on the film. The lane in which that band appeared iden- Polyacrylamide gels from 6% to 20% are used for
tified the nucleotide. Bands in the purine (G + A) or sequencing. Bromophenol blue and xylene cyanol
loading dyes are used to monitor the migration of
T
3′ the fragments. Run times range from 1 to 2 hours
G G+A C+T C G for short fragments (up to 50 base pairs [bp])
C to 7 to 8 hours for longer fragments (more than
T
T
150 bp).
T
A
G Dideoxy Chain Termination (Sanger) Sequencing
A
A Dideoxy chain termination (Sanger) sequencing is a
T
A
modification of the DNA replication process. A short,
T synthetic, single-stranded DNA fragment (primer) com-
C plementary to sequences just 5′ to the region of DNA to
G be sequenced is used for priming dideoxy sequencing
A
G reactions (Fig. 9.3). For detection of the products of the
C sequencing reaction, the primer is attached covalently at
A
T Primer
G 5′ –3′ OH
C
3′ … T C G A C G G G C … 5′
C
A Template
5′ Area to be
sequenced
FIGURE 9.2 Products of a Maxam–Gilbert sequencing reac-
tion. The gel is read from the bottom to the top. The size of the FIGURE 9.3 Manual dideoxy sequencing requires a sin-
fragments gives the order of the nucleotides. The nucleotides gle-stranded version of the fragment to be sequenced (tem-
are inferred from the lane in which each band appears. A or T plate). Sequencing is primed with a short synthetic piece of
is indicated by bands that appear in the G + A lane or C + DNA complementary to bases just before the region to be
T lane, respectively, but not in the G lane or the C lane. G is sequenced (primer). The sequence of the template will be
present in the G + A lane and the G lane. C is present in the determined by extension of the primer in the presence of
C + T lane and the C lane. dideoxynucleotides.
Nitrogen base Nitrogen base and translation machinery to make new sin-
gle-stranded genomes and viral proteins. To use
HOCH2 O HOCH2 O M13 for template preparation, the RF was iso-
C C C C lated from infected bacteria, cut with restriction
C C C C enzymes, and the fragment to be sequenced was
OH H H H
ligated into the RF. When the recombined RF was
reintroduced into the host bacteria, M13 contin-
dNTP ddNTP ued its life cycle producing new phages, some of
FIGURE 9.4 A dideoxynucleotide (right) lacks the hydroxyl which carried the inserted fragment. When the
group on the 3′ ribose carbon that is required for formation of phages were spread on a lawn of host bacteria,
a phosphodiester bond with the phosphate group of another plaques (clear spaces) of lysed bacteria formed
nucleotide. by phage replication contained pure populations
of recombinant phage. The single-stranded DNA
the 5′ end to a 32P-labeled nucleotide or a fluorescent was then isolated from the phage by picking
dye-labeled nucleotide. A previously used alternative plugs of agar from the plaques and isolating DNA
detection strategy was to incorporate 32P- or 35S-labeled from them.
deoxynucleotides in the nucleotide sequencing reaction
mix (internal labeling).
Just as in the in vivo DNA replication reaction, an
in vitro DNA synthesis reaction would result in poly-
Advanced Concepts
merization of deoxynucleotides to make full-length
An advantage of the M13 template preparation
copies of the DNA template. For sequencing, modified
method was that the primer that hybridizes to M13
dideoxynucleotide (ddNTP) derivatives are added
sequences could be used to sequence any fragment
to the reaction mixture. Dideoxynucleotides lack the
ligated into the same site of the RF. Recombinant
hydroxyl group found on the 3′ ribose carbon of the
plasmids containing fragments to be sequenced
deoxynucleotides (dNTPs; Fig. 9.4). DNA synthesis will
include a short M13 region so that the M13 uni-
stop upon incorporation of a ddNTP into the growing
versal primer could still be used in some appli-
DNA chain (chain termination) because without the
cations, even though the M13 method of template
hydroxyl group at the 3′ sugar carbon, the 5′–3′ phos-
preparation is no longer practical.
phodiester bond cannot be established to incorporate a
subsequent nucleotide. The newly synthesized chain will
terminate, therefore, with the ddNTP (Fig. 9.5). For manual dideoxy sequencing, a 1:1 mixture of tem-
plate and radioactively labeled primer is placed into four
separate reaction tubes in sequencing buffer contain-
Histooricaal Higghlligghtts ing the sequencing enzyme and ingredients necessary
for the polymerase activity (Fig. 9.6). Mixtures of all
The original dideoxy chain termination sequenc-
four dNTPs and one of the four ddNTPs are then added
ing methods used in the late 1970s into the early
to each tube, with a different ddNTP in each of the
1980s required a single-stranded template. Tem-
four tubes.
plates up to a few thousand bases long were
produced using M13 bacteriophage, a bacterial
virus with a single-stranded DNA genome. This Advanced Concepts
virus replicates by infecting Escherichia coli, in
which the viral single-stranded circular genome Polymerase chain reaction (PCR) products are
is converted to a double-stranded plasmid, the currently used as sequencing templates. Resid-
replication factor (RF). The plasmid codes for ual components of the PCR reaction, especially
viral gene products use the bacterial transcription
Growing strand
O– P O O– P O
Template strand
O O
H2C O H2C O
A T A T
CH CH2 CH CH2
HC CH2 HC CH2
O O
O– P O O– P O
O O
H2C O H2C O
G C G C
CH CH2 CH CH2
HC CH2 HC CH2
OH
O O O O O O
– –
O P O P O P O– O P O P O P O–
O– O– O C G O– O– O C G
H2C O H2C O
CH CH CH CH
HC CH2 HC CH2
OH OH
FIGURE 9.5 DNA replication (left) is terminated by the absence of the 3′ hydroxyl group on the dideoxyguanosine nucleotide
(ddG, right). The resulting fragment ends in ddG.
is too high, polymerization will terminate too frequently

primers and nucleotides, can interfere with the early along the template. If the ddNTP concentration is
sequencing reaction and lower the quality of the too low, infrequent or no termination will occur. In the
sequencing ladder. PCR amplicons can be cleaned beginning days of sequencing, optimal ddNTP/dNTP
using solid-phase (column or bead) matrices, ratios were determined empirically (by experimenting
alcohol precipitation, or enzymatic digestion with with different ratios). Sequencing reagent mixes have
alkaline phosphatase. Alternatively, amplicons can preoptimized nucleotide mixes.
be run on an agarose gel and the bands eluted. With the addition of DNA polymerase enzyme to the
The latter method provides not only a clean tem- four tubes, the reaction begins. After about 20 minutes,
plate but also confirmation of the product being the reactions are terminated by addition of a stop buffer,
sequenced. It is especially useful when the PCR which consists of 20 mM EDTA to chelate cations and
reactions are not completely free of mis-primed stop enzyme activity, formamide to denature the prod-
bands or primer dimers. ucts of the synthesis reaction, and gel loading dyes (bro-
mophenol blue and/or xylene cyanol). All four reactions
are carried out for equal times to provide consistent
The ratio of ddNTPs/dNTPs is critical for the generation band intensities in all four lanes of the sequencing gel
of a readable sequence. If the concentration of ddNTPs sequence.
The sets of synthesized fragments are then loaded onto

ddATP + four dNTPs ddA
a denaturing polyacrylamide gel. The products of each
A dAdGdCdTdGdCdCdCdG of the four sequencing reactions are loaded into adja-
cent lanes, labeled A, C, G, or T, corresponding to the
ddNTP in the four reaction tubes. Once the gel is dried
and exposed to x-ray film, the fragment patterns are
ddCTP + four dNTPs dAdGddC visualized by the signal on the 32P-labeled primer (or
C dAdGdCdTdGddC
dAdGdCdTdGdCddC
incorporated deoxynucleotide). All fragments from a
dAdGdCdTdGdCdCddC given tube will end in the same ddNTP; for example, all
the fragments synthesized in the ddCTP tube end in C.
ddGTP + four dNTPs dAddG The four-lane gel electrophoresis pattern of the prod-
G dAdGdCdTddG ucts of the four sequencing reactions is called a sequenc-
dAdGdCdTdGdCdCdCddG ing ladder (Fig. 9.7). The ladder is read to deduce the
DNA sequence. From the bottom of the gel, the smallest
dAdGdCddT
(fastest-migrating) fragment is the one in which synthe-
ddTTP + four dNTPs
T dAdGdCdTdGdCdCdCdG sis terminated closest to the primer. The identity of the
ddNTP at a particular position is determined by the lane
in which the band appears. If the smallest band is in the
FIGURE 9.6 Components required for DNA synthesis (tem- ddATP lane, then the first base is an A. The next larger
plate, primer, enzyme, buffers, dNTPs) are mixed with a differ- fragment is the one that was terminated at the next posi-
ent ddNTP in each of four tubes (left). With the proper ratio of
tion on the template. The lane that has the next larger
ddNTPs/dNTPs, the newly synthesized strands of DNA will
terminate at each opportunity to incorporate a ddNTP. The
band identifies the next nucleotide in the sequence.
resulting synthesis products are a series of fragments ending in The sequence is thus read from the bottom (smallest,
either A (ddATP), C (ddCTP), G (ddGTP), or T (ddTTP). This 5′-most) to the top (largest, 3′-most) fragments across
collection of fragments is the sequencing ladder. or within lanes to determine the identity and order of
nucleotides in the sequence.
Depending on the reagents and gel used, the number of
bases per sequence read averages 300 to 400. Advances
in enzyme and gel technology have increased this capa-
Advanced Concepts bility to over 500 bases per read. Sequencing reads are
lengthened by loading the same ladders in intervals of
Manganese (Mn++) added to the sequencing reac- 2 to 6 hours so that the larger bands are resolved with
tion promotes equal incorporation of all dNTPs longer (e.g., 8-hour) migrations, whereas smaller bands
by the polymerase enzyme. Equal incorporation will be resolved simultaneously in a 1- to 2-hour migra-
of the dNTPs makes for uniform band intensities tion that was loaded 6 to 7 hours later.
on the sequencing gel, which eases interpretation As Sanger sequencing came into routine use, technol-
of the sequence. Manganese increases the rela- ogy was improved significantly from these first manual
tive incorporation of ddNTPs as well, which will sequencing procedures. Recombinant polymerase
enhance the reading of the first part of the sequence enzymes with in vitro removal of the exonuclease activ-
by increasing intensity of the smaller bands on ity were faster and more processive (i.e., they stayed
the gel. Modified nucleotides, deaza-dGTP and with the template longer, producing longer sequencing
deoxyinosine triphosphate (dITP), are also added ladders). In addition, these engineered enzymes more
to sequencing reaction mixes to deter secondary efficiently incorporated ddNTPs and nucleotide analogs
structure in the synthesized fragments. Such addi- such as dITP or deaza-dGTP, which were used to deter
tives as Mn++, deaza-dGTP, and dITP are supplied secondary structure (internal folding and hybridiza-
in commercial sequencing buffers. tion) in the template and sequencing products. Further-
more, sequencing was performed with double-stranded
A C T G A C T G
3′
G
T 3′
C
A
A
C Gel area more
T difficult to read
G
A
A
T
C
C
C
T
FIGURE 9.7 A sequencing ladder is read from G
the bottom of the gel to the top. The smallest C
G
(fastest-migrating) fragment represents the first A 5′
nucleotide attached to the primer by the poly- 5′
merase. Since that fragment is in lane A, from the
reaction that contained ddATP (left), the sequence
5′ AGCGTCCCTAAGTCAACTG 3′
read begins with A. The next largest fragment is
in lane T. The sequence, then, reads AT. The next
largest fragment is in lane C, making the sequence
ATC, and so forth up the gel. Larger bands on a
sequencing gel can sometimes be compressed,
limiting the length of sequence that can be read
on a single gel run (right).
templates, eliminating the requirement for the preparation early high-throughput applications and automation. Uni-
of single-stranded versions of the DNA to be sequenced. versal systems combined automation of DNA isolation
Using heat-stable enzymes, the sequencing reaction of the template and setup of the sequencing reactions.
took place in a thermal cycler (cycle sequencing). With Electrophoresis and reading of the sequencing ladder
cycle sequencing, timed manual starting and stopping of were also automated. A requirement for automated
the sequencing reactions were not necessary. The labor reading of the DNA sequence ladder is the use of flu-
savings in this regard increase the number of reactions orescent dyes instead of radioactive nucleotides to label
that could be performed simultaneously; for example, the primers or sequencing fragments.
a single operator could run 96 sequencing reactions
(i.e., sequence 24 fragments) in a 96-well plate. Finally, Advanced Concepts
improvements in fluorescent dye technology have led to
the automation and throughput of the sequencing process Fluorescent dyes used for automated sequenc-
and, more importantly, sequence determination. ing include fluorescein, rhodamine, and Bodipy
(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dye
derivatives that are recognized by commercial
Automated Fluorescent Sequencing
detection systems.4 Automated sequence readers
The chemistry for automated sequencing is the same excite the dyes with a laser and detect the emit-
as that described for manual sequencing, using double- ted fluorescence at specific wavelengths. More ad-
stranded templates and cycle sequencing. Because cycle vanced methods have been proposed to enhance
sequencing (unlike manual sequencing) does not require the distinction between the dyes for more accurate
the sequential addition of reagents to start and stop the determination of the sequence.5
reaction, cycle sequencing was more easily adaptable to
G A T C ddCTP, ddGTP, or ddTTP, as shown in Figure 9.9. After

addition of the remaining components of the sequencing
G reaction (see the previous section on manual sequencing)
T and of a heat-stable polymerase, the reaction is subjected
C to cycle sequencing in a thermal cycler. The products of
T the sequencing reaction are then labeled at the 5′ end,
G using the dye color associated with the ddNTP at the end
A
of the fragment.
Dye terminator sequencing is performed with one
Gel electrophoresis
of the four fluorescent dyes covalently attached to each
Capillary electrophoresis of the ddNTPs instead of to the primer. The primer is
FIGURE 9.8 Instead of four gel lanes (left) fluorescent frag- unlabeled. A major advantage of this approach is that
ments can be run in a single gel lane or in a capillary (right). all four sequencing reactions are performed in the same
Note that the sequence of nucleotides, AGTCTG, read by lane tube (or well of a plate) instead of in four separate tubes.
in the slab gel is read by color in the capillary. After addition of the rest of the reaction components and
cycle sequencing, the product fragments are labeled at
the 3′ end. As with dye primer sequencing, the “color”
Fluorescent dyes used for sequencing have distinct
of the dye corresponds to the ddNTP that terminated
“colors,” or peak wavelengths of fluorescence emission,
the strand. Dye terminator sequencing has become the
that can be distinguished by automated sequencers. The
Sanger sequencing method of choice. The option of one
advantage of having four distinct colors is that all four
reaction for all four nucleotides lowers the cost and labor
of the reaction mixes can be read in the same lane of a
of routine sequencing performed in many laboratories.
gel or on a capillary. Fluorescent dye color rather than
lane placement will assign the fragments as ending in A,
T, G, or C in the sequencing ladder (Fig. 9.8). The Sequencing Ladder
After a sequencing reaction using fluorescent dye ter-
Approaches to Automated Sanger Sequencing
minators, excess dye terminators are removed with
There are two approaches to automated fluorescent columns or beads or by ethanol precipitation. Spin
sequencing: dye primer and dye terminator sequenc- columns or bead systems bind the sequencing fragments
ing (Fig. 9.9). The goal of both approaches is to label to allow removal of residual sequencing components by
the fragments synthesized during the sequencing reac- rinsing with buffers. Alternately, the dye terminators are
tion according to their terminal ddNTP. Thus, frag- bound onto specially formulated magnetic beads, and
ments ending in ddATP, read as A in the sequence, will the sequencing ladder is recovered from the supernatant
be labeled with a “green” dye; fragments ending in as the beads are held by a magnet applied to the outside
ddCTP, read as C in the sequence, will be labeled with of the tube or plate.
a “blue” dye; fragments ending in ddGTP, read as G in The fragments of the sequencing ladder are com-
the sequence, will be labeled with a “black” or “yellow” pletely denatured before running on a gel or capillary.
dye; and fragments ending in ddTTP, read as T in the Denaturing conditions (50°C to 60°C, formamide, urea
sequence, will be labeled with a “red” dye. This facili- denaturing gel) are maintained so that the fragments are
tates reading of the sequence by the automated sequence. resolved strictly according to size. Secondary structure
In dye primer sequencing, the four different fluores- affects migration speed and lowers the quality of the
cent dyes are attached to four separate aliquots of the sequence. Before loading in a gel or capillary instru-
primer. The dye molecules are attached covalently to the ment, sequence ladders are cleaned, as described previ-
5′ end of the primer during chemical synthesis, resulting ously, to remove residual dye terminators, precipitated,
in four versions of the same primer with different dye and resuspended in formamide. The ladders are heated
labels. The primer labeled with each “color” is added to 95°C to 98°C for 2 to 5 minutes and placed on ice
to four separate reaction tubes, one each with ddATP, just before loading.
Automated dye primer sequencing Automated dye terminator sequencing
Dye primer Primer
A
A Dye terminators
ACCGTA
ddATP ACCGTA AC
ddATP Dye
terminator ACCGTAT
ddCTP ACC
removal
ddGTP ACCG Completed
ACCGT sequencing
ddTTP reaction
ACCGTAT
AC
ddCTP ACC
Ethanol
ACCGTAT
precipitation
Completed
sequencing
reaction
ddGTP ACCG
ACCGT
ddTTP ACCGTAT
FIGURE 9.9 Fluorescent sequencing chemistries. Dye primer sequencing uses labeled primers (left). The reactions take place in
separate tubes and the products of all four reactions are resolved together in one lane of a gel or in a capillary. Using dye termina-
tors (right), only one reaction tube is necessary because the fragments can be distinguished directly by the dideoxynucleotides on
their 3′ ends.
attached to each fragment, causing the dye to emit flu-

Electrophoresis orescence that is captured by the detector. The detector
The four sets of sequencing products in each reaction are converts the fluorescence to an electrical signal that is
loaded onto a single gel lane or capillary. The fluores- imaged by computer software as a flash or peak of color.
cent dye colors, rather than lane assignment, distinguish
which nucleotide is at the end of each fragment. Running Advanced Concepts
all four reactions together not only increases throughput
but also eliminates lane-to-lane migration variations that DNA sequences with high GC content are some-
affect the accurate reading of the sequence. The migrat- times difficult to read due to intrastrand hybrid-
ing fragments pass a laser beam and a detector in the ization in the template DNA. Reagent preparations
automated sequencer. The laser beam excites the dye
and the cleanliness of the sequencing ladder. Failure to

that include 7-deaza-dGTP (7-Deaza-2′-deoxy- clean the sequencing ladder properly results in bright
guanosine-5′-triphosphate) or dITP instead of flashes of fluorescence (dye blobs) that obliterate parts
standard dGTP improve the resolution of bands of the sequence read (Fig. 9.10). Poor starting material
(peaks) in regions that exhibit GC band compres- results in a poor-quality sequence that cannot be read
sions, or bunching of peaks close together so that accurately (Fig. 9.11). Clear, clean sequencing ladders
they are not resolved, followed by several peaks are read accurately by the software, and a text sequence
running farther apart. is generated. Sequencing software also shows the cer-
tainty of each base call in the sequence. When the base
call is not clear, the letter N will replace A, C, T, or G.
Fluorescent detection equipment yields results as an elec- Less-than-optimal sequences are not accurately read-
tropherogram, rather than a gel pattern. Just as the gel able by software but may be readable by an experienced
sequence is read from the smallest (fastest-migrating) operator.
fragments to the largest, the sequencing software reads, Software programs can compare two sequences or test
or “calls,” the bases from the smallest (fastest-migrating) sequences with reference sequences to identify mutations
fragments that first pass the detector to the largest based or polymorphisms. Regardless of whether a sequence
on the dye emission wavelength; that is, the software calls variant (change from a reference sequence) is found, it
the base by the “color” of the fluorescence of the frag- is important to sequence both complementary strands of
ment as it passes the detector. The electropherogram is a DNA to confirm sequence data. This is especially criti-
series of peaks of the four fluorescent dyes as the bands cal for confirmation of mutations or polymorphisms in a
of the sequencing ladder migrate by the detector. The sequence (Fig. 9.12). Alterations affecting a single base
software assigns one of four colors—red, black, blue, or pair may be subtle on an electropherogram, especially if
green—associated with each of the fluorescent dyes and the alteration is in the heterozygous form, or mixed with
a text letter to the peaks for ease of interpretation. the normal reference sequence. Ideally, a genetically het-
As with manual sequencing, the ratio of ddNTPs/ erozygous mutation appears as two peaks of equal height
dNTPs is key to the length of the sequence read (how but different colors directly on top of one another, that is,
much of the template sequence can be determined). Too at the same position in the electropherogram. The over-
many ddNTPs will result in a short sequence read. Too lapping peaks should be about half the height of single
low a concentration of ddNTPs will result in loss of base peaks. Heterozygous deletions or insertions (e.g.,
sequence data close to the primer but give a longer read the BRCA frameshift mutations) affect all positions of
because the sequencing enzyme will polymerize further the sequence downstream of the mutation (Fig. 9.13).
down the template before it incorporates a ddNTP into Somatic mutations in clinical specimens are sometimes
the growing chain. The quality of the sequence (height difficult to detect because they may be diluted by normal
and separation of the peaks) improves away from the sequences that mask the somatic change.
primer and begins to decline at the end. At least 400 Several software programs have been written to
to 500 bases can be easily read with most sequencing interpret and apply sequence data from capillary elec-
chemistries. trophoresis. Software that collects the raw data from the
instrument is supplied with the electrophoresis instru-
ments. Software that interprets, compares, or otherwise
Sequence Interpretation
manipulates sequence data is sometimes supplied with a
Base calling is the process of identification of bases in purchased instrument or available online. A representa-
a sequence by sequencing software. It is analogous to tive sample of these applications is shown in Table 9.2.
the inspection of gel bands for quality, clarity, and sep- Further sequence interpretation with regard to disease
aration. Interpretation of sequencing data from a dye association and pathogenic significance requires the use
terminator reaction depends on the quality of the elec- of sequence databases and clinical trial information.
tropherogram, which, in turn, depends on the quality of This information is available from public websites and
the template, the efficiency of the sequencing reaction, institutional “data commons” collections.
A
C
G
T
C C T T T T T G A A A T A A A G N C C T G C C C N G T A T T G C T T T A A A C A A G A T T T
10 20 30 40
C C T C T A T T G T T G G A T C A T T C G T C A C A A A A T G A T T C T G A A T T A G C G T A T C G T
60 70 80 90 100
FIGURE 9.10 Electropherogram showing a dye blob at the beginning of a sequence (nucleotide positions 9 to 15). The sequence
read around this area is not accurate. See Color Plate 5.
A
C
G
T
G A T T C T G A A T T A G C T G T A T C G N N T T S T G N M A T Y N K C T K N A T C G
FIGURE 9.11 Examples of good sequence quality (left) and poor sequence quality (right). Note the clean baseline on the good
sequence; that is, only one color peak is present at each nucleotide position. Automatic sequence reading software will not accu-
rately call a poor sequence. Compare the text sequences below the two scans. See Color Plate 6.
A
C
G
T
G C T G G T G G C G T A G C T T G T G G C G T A G C T A C G C C A C A A G C
G C
FIGURE 9.12 Sequencing of a heterozygous G to T mutation in exon 12 of the KRAS gene. The normal codon sequence is GGT
(left). The heterozygous mutation (GT, center) is confirmed in the reverse sequence (CA, right). See Color Plate 7.
A
C
G
T
G T A T G C A G A A A A T C T T A G A G T G T C C C A T C T G G T A A G T C A G C
G T A T G C A G A A A A T C T T A G W G T S T C M Y M T S K K G R W A W S T S M R C
FIGURE 9.13 The 187 delAG mutation in the BRCA1 gene detected by Sanger sequencing. This heterozygous dinucleotide dele-
tion is evident in the lower panel where, at the site of the mutation, two sequences are overlaid: the normal sequence and the
normal sequence minus two bases. See Color Plate 8.
TABLE 9.2 Examples of Software Programs Used to Analyze and Apply Sequence Data
Software Name Application
BLAST Basic Local Alignment Search Tool Compares an input sequence with all sequences in a selected
database
GRAIL Gene Recognition and Assembly Internet Link Finds gene-coding regions in DNA sequences
FASTA FAST-All derived from FAST-P (protein) and Rapidly aligns pairs of sequences by sequence patterns rather
FASTQ FAST-N (nucleotide) search algorithms than individual nucleotides
Biological data with quality score
Phred Phred Reads bases from original trace data and recalls the bases,
assigning quality values to each base
Polyphred Polyphred Identifies single-nucleotide polymorphisms (SNPs) among the

traces and assigns a rank indicating how well the trace at a site
matches the expected pattern for an SNP
Phrap Phragment Assembly Program Uses user-supplied and internally computed data quality
information to improve accuracy of assembly in the presence
of repeats
TIGR The Institute for Genomic Research Developed by TIGR as an assembly tool to build a consensus
Assembler sequence from smaller-sequence fragments
Factura Factura Identifies sequence features such as flanking vector sequences,

restriction sites, and ambiguities
SeqScape SeqScape Provides mutation and SNP detection and analysis, pathogen
subtyping, allele identification, and sequence confirmation
Assign Assign Identifies alleles for haplotyping
Matchmaker Matchmaker Identifies alleles for haplotyping
PYROSEQUENCING sulfurylase, and luciferase, plus the two substrates ade-

nosine 5′ phosphosulfate (APS) and luciferin. One of
Chain termination sequencing became the most widely the four dNTPs is added in a predetermined order to the
used method to determine DNA sequence. Other reaction. If the nucleotide is complementary to the base
methods were developed that yielded the same infor- in the template strand next to the 3′ end of the primer,
mation but with less throughput capacity than the chain DNA polymerase extends the primer. Pyrophosphate
termination method. Pyrosequencing is an example of a (PPi) is released with the formation of the phosphodi-
method designed to determine a DNA sequence without ester bond between the dNTP and the primer. The PPi is
having to make a sequencing ladder.6,7 This procedure converted to ATP by sulfurylase that is used to generate
relies on the generation of light (luminescence) when a luminescent signal by luciferase-catalyzed conversion
nucleotides are added to a growing strand of DNA of luciferin to oxyluciferin.
(Fig. 9.14). With this system, there are no gels, fluores- The process is repeated with each of the four nucle-
cent dyes, or ddNTPs. otides again added sequentially to the reaction. The
The pyrosequencing reaction mix consists of a generation of a signal indicates which nucleotide is the
single-stranded DNA template, sequencing primer, next correct base in the sequence. The results from a
Step 1
Polymerase
(DNA)n + dNTP (DNA)n+1 + PPi
Step 2
Luciferin Oxyluciferin
Sulfurylase
Luciferase
APS + PPi
Light
ATP Light
Time
Step 3
Apyrase
nNTP dNDP + dNMP + phosphate
Apyrase
ATP ADP + AMP + phosphate
Nucleotide sequence
FIGURE 9.14 Pyrosequencing is the analysis of
G C – A GG CC T pyrophosphate (PPi) released when a nucleotide
base (dNTP) is incorporated into DNA (top left).
The released PPi is a cofactor for ATP generation
from adenosine 5′ phosphosulfate (APS). Lucifer-
ase plus ATP converts luciferin to oxyluciferin with
the production of light, which is detected by a lumi-
nometer. The system is regenerated with apyrase
that degrades residual free dNTP and dATP
(Step 3). As nucleotides are added to the system
G C T A G C T
one at a time, the sequence is determined by which
Nucleotide added of the four nucleotides generates a light signal.
pyrosequencing reaction, a pyrogram, consist of peaks polymorphism (SNP) and typing (re-sequencing) rather
of luminescence associated with the addition of the com- than for generating new sequences. It has been used for
plementary nucleotide (Fig. 9.14). If a sequence con- applications in mutation detection,8 infectious disease
tains a repeated nucleotide, for instance, GCAGGCCT, typing,9,10 and DNA methylation analysis.11
the results would be dG peak, dC peak, dA peak, dG
peak (double height), dC peak (double height), dT peak.
The nucleotide sequence is called based on the order of BISULFITE DNA SEQUENCING
nucleotide bases introduced to the sequencing reaction
and the peak heights. Bisulfite DNA sequencing, or methylation-specific
Pyrosequencing is most useful for short- to moderate- sequencing, is chain termination sequencing designed
sequence analysis. It is therefore used mostly for detec- to detect methylated cytosine nucleotides.12 Methylation
tion of previously known mutation or single-nucleotide of cytosine residues to 5-methylcytosines in DNA is an
important part of the regulation of gene expression and The PCR amplicons are then sequenced by Sanger
chromatin structure, affecting cell differentiation and sequencing or pyrosequencing. Methylation is detected
diseases, including several types of cancer. by comparing the treated sequence with the original
For bisulfite sequencing, 2 to 4 μg of genomic DNA sequence (before conversion) and noting where in the
is cut with restriction enzymes to facilitate denatur- treated sequence cytosines are not changed to thymine
ation. The enzymes should not cut within the region (uracil); that is, the converted sequence will be altered
to be sequenced. The restriction digestion products are relative to the reference sequence at the unmethylated
resolved on an agarose gel, and the fragments of the size C residues.
of interest are purified from the gel. DNA from fixed In Sanger sequencing, unmethylated cytosines will
tissue may be used directly without restriction digestion. appear as red (thymine) instead of blue (cytosine) peaks
The DNA is denatured with heat (97°C for 5 minutes) on the electropherogram. In pyrosequencing, the relative
and exposed to bisulfite solution (sodium bisulfite, light intensity of consecutive T and C additions to the
NaOH, and hydroquinone) for 16 to 20 hours. Buffer reaction mix provide a quantitative degree of methyl-
systems that protect DNA from bisulfite damage may ation. An example of pyrosequencing of bisulfite con-
be used to increase the yield of converted DNA. Over- verted DNA is shown in Figure 9.15, where the color
exposure to bisulfite can result in strand cleavage and or height of the cytosine peaks relative to the thymine
loss of important regions of the DNA template. During (uracil) peaks indicates the degree of methylation.
the incubation with bisulfite, the cytosines in the reac- Detection methods other than sequencing have also
tion are deaminated, converting them to uracils, whereas been devised to detect DNA methylation, such as using
the 5-methylcytosines are unchanged. methylation-sensitive restriction enzymes or enzymes
with recognition sites generated or destroyed by the C
to U changes. Other methods use PCR primers that will
Advanced Concepts bind only to the converted or nonconverted sequences
so that the presence or absence of PCR product indi-
Pyrosequencing requires a single-stranded cates the methylation status. These methods, however,
sequencing template. Methods using streptavi- are not always applicable to the detection of methylation
din-conjugated beads have been devised to easily in unexplored sequences. As the role of methylation and
prepare the template. First the region of DNA to epigenetics in human disease is increasingly recognized,
be sequenced is PCR-amplified with one of the bisulfite sequencing has become a popular method in the
PCR primers covalently attached to a biotin mol- research laboratory. Clinical tests have been developed
ecule. The double-stranded amplicons are then using this strategy as well.13,14
immobilized onto the beads and denatured with
NaOH. After several washings to remove the
non-biotinylated complementary strand (and all RNA SEQUENCING
other reaction components), the sequencing primer
is added and annealed to the pure single-stranded The sequences of RNA transcripts are, for the most
DNA template. part, complementary to their DNA templates. Post-
transcriptional processing of RNA, however, changes
the RNA sequence relative to its encoding DNA. Alter-
After the reaction, the treated DNA is cleaned, precipi- native splicing and RNA editing may further modify the
tated (or purified by adhering and washing on columns RNA sequence. Early methods to sequence RNA made
or beads), and resuspended for use as a template for use of ribonucleases to cut end-labeled RNA at spe-
PCR amplification. The primers used for amplifica- cific nucleotides. Another approach was to infer mRNA
tion are altered to accommodate C to U changes in the sequence from amino acid sequence. The RNA transcript
primer-binding sites caused by the bisulfite treatment. sequence can be determined from the sequencing of its
For pyrosequencing, one primer is biotinylated for isola- complementary DNA; however, sequencing error may
tion of the single-stranded template. occur, mostly from the cDNA synthesis step.15,16
C5 : YGYGTTTATGYGAGGTYGGGTGGGYGGGTYGTTAGTTTYG
0% 0% 1% 0% 1% 1% 1%
1200
1000
800
600
400
200
0
–200
E S G T C T G T C G T A T A G T C G A T G T C G T A G T C T G T C G T A T G T T C
5 10 15 20 25 30 35
A4 : YGYGTTTATGYGAGGTYGGGTGGGYGGGTYGTTAGTTTYG
37% 1% 33% 38% 42% 46% 46%
1500
1000
500
E S G T C T G T C G T A T A G T C G A T G T C G T A G T C T G T C G T A T G T T C
5 10 15 20 25 30 35
FIGURE 9.15 DNA methylation at cytosine residues detected by pyrosequencing of bisulfite-treated DNA. Exposure of a sequence
to bisulfite will result in the conversion of unmethylated cytosines to uracils (T in the sequence). The pyrosequencing method will
report the percent methylation that is the relative number of C to T nucleotides at each potentially methylated C position (shaded).
The C residues in the top panel are not methylated. All but one of the C residues in the bottom panel are methylated.
Direct sequencing of RNA has been proposed based

on single-molecule sequencing technology and virtual NEXT-GENERATION SEQUENCING
terminator nucleotides.17,18 In this method, mRNA is cap-
tured by immobilized polydT oligomers (Fig. 9.16). For Data obtained from sequence analysis is best inter-
those RNA species without polyA tails, an initial treat- preted in context with population norms and variations;
ment with polyA polymerase is performed to add a 3′ however, initially, few large sequence analyses were
A-tail. The 3′ ends of the captured RNA are chemically performed for multiple individuals. Furthermore, disease
blocked to prevent extension in the sequencing step. states involve a variety of sequence variants that can be
Four reversibly dye-labeled nucleotides are then sequen- important for diagnosis, prognosis, and treatment strat-
tially added. An image is taken, the extension inhibitors egy. Although array studies were applied to this type of
are cleaved, and alternating C, T, A, or G nucleotides analysis, even the densest oligo array did not provide
are added, with imaging, cleavage, and rinsing between genomic-scale sequence data with single-base-pair res-
each nucleotide addition. After repeating this process olution. Next-generation sequencing (NGS), also called
many times (e.g., 120 cycles) the collected images massive parallel sequencing, was designed to sequence
are aligned and used to build the sequence from each large numbers of templates carrying millions of bases
poly(dT) anchor. simultaneously, in a run that takes a few hours. NGS
End repair
A
A-tailing
A
FIGURE 9.16 A next-generation sequencing T

library is created by the fragmentation of DNA. The T
fragmented DNA may have single-stranded ends that T
must be repaired back to double-stranded blunt ends Adapters
T
by end repair. Addition of an A residue on the
repaired blunt ends facilitates ligation of adapters
carrying primer-binding sites for PCR amplification T T
of the library. Index Tail
technology has achieved gigabytes of sequencing data single-molecule capabilities.19 Powerful computer data
for a minimal cost, making genomic studies a routine assembly systems are required to organize the massive
component of both research and clinical analysis. amounts of sequence information that are generated.
These technologies can be used not only to sequence
whole genomes but also to investigate populations of
Histooricaal Higghlligghtts small genomes such as microbial diversity.20
Among the early challenges with massive sequencing
Early studies of DNA polymerase activity on an was the integration of technologies without compromis-
immobilized template led to the development of ing accuracy or throughput.21,22 These issues have been
multiple template arrays that could be sequenced addressed with advances in bioinformatics and computer
simultaneously. In 1997 high-throughput Selexa software. New challenges with system design, data accu-
technologies were designed with capabilities mulation and storage, clinical sensitivity, and data inter-
of whole genome sequencing. High-throughput pretation are being addressed, especially in dedicated
sequencing platforms developed in the mid- sequencing facilities and commercial bioinformatics
2000s resulted in a 50,000-fold drop in the cost services.
of human genome sequencing from that of the NGS requires strong computer support as well as tera-
Human Genome Project and led to the term next- bytes of storage space to accommodate large raw data
generation sequencing. These technologies have sets. To prepare for NGS, clinical laboratories estab-
increased in capacity and have been refined to lish secure information channels and allocate space for
address sequence complexity in genomes. The preparation, loading, and operation of the sequencers.
cost of sequencing a human genome has achieved Interface with laboratory information systems and elec-
the $1,000 cost point, which has expanded the use tronic medical records might also be arranged. Report
of sequencing analyses in the clinical laboratory. templates are designed by the laboratory or commercial
vendors and bioinformatics services.23
Two NGS technologies account for the majority
NGS technologies include pyrosequencing, reversible of clinical sequencing applications: ion-conductance
dye terminator sequencing, ion-conductance sequenc- (pH)24 and reversible dye terminator sequencing.25 Both
ing, single-molecule sequencing, and sequencing by methods require the preparation of a sequencing library,
ligation. NGS requires novel methods of template sets of 100- to 500-bp-size fragments representing the
preparation, such as emulsion PCR and bridge PCR, or regions to be sequenced. A library can represent a whole
genome or a few specific gene regions where critical NGS Library Preparation
variants are likely to occur.
A collection of DNA fragments to be sequenced is a
sequencing library. Reversible dye terminator and
Gene Panels ion-conductance sequencing are performed on DNA
The size and application of the sequencing library depend fragments less than 1,000 bp in length. Genomic DNA
on the selection of genes to be sequenced or gene panels. is fragmented by a number of methods, including shear-
Gene panels are probe or primer sets designed to amplify ing with high-frequency acoustic energy, sonication,
specific genes, regions, or entire exomes (all protein-cod- nebulization (forcing DNA molecules through a small
ing sequences).26 NGS might also be performed to opening), or enzymatic treatments. Particular methods
compare sequences of many organisms (rRNA genes in and how they are used (e.g., pressure levels used in neb-
microbial speciation) or to detect large numbers of pos- ulization) produce differently sized fragments (100 to
sible base differences in a highly polymorphic gene such 1,000 bp). The median fragment size can be checked by
as CFTR. Gene panels have high technical sensitivity but gel electrophoresis or microfluidics. Starting DNA con-
require knowledge of the clinical diagnosis that would centrations and the DNA concentration of the library is
justify testing particular genes. best measured by fluorometry.
Gene panels have been designed for disease states,
such as cardiomyopathies or muscular dystrophy or Advanced Concepts
cancers. These panels range from a few (less than 20)
target genes to more than a thousand target genes such Sequencing protocols and technologies differ with
as those used for solid organ cancers. “Hot-spot” panels respect to the amount of required input genomic
target regions of specific genes known to affect treatment DNA. The lower limits range from 10 to 50 ng
response, disease state, or clinical condition. Variants of DNA. For sequencing tumor DNA from fixed
in these regions are referred to as “actionable” muta- tissue, 140 mm2 tissue with at least 30% tumor
tions; that is, a therapeutic or medical measure might be is recommended. Suboptimal amounts of start-
taken as a result of the presence of a variant. Targeted ing DNA will compromise sequence quality and
panels include critical genes in particular diseases such increase the risk of PCR artifacts. Fluorometric
as hematological-cancer-specific panels for lymphoid measurement of input (and library) concentrations
or myeloid disorders or solid-tumor-specific panels for is recommended over spectrometry to ensure the
lung, colon, breast, or other cancers. Very large panels measurement of intact DNA.
up to 3,000 genes or more provide a large amount of
information for diagnostic, prognostic, and discovery
purposes. These panels, however, may produce vari- Fragmented DNA produced by enzymatic or physi-
ants of unknown significance that must be assessed by cal methods may be used directly for whole-exome or
pathologists and oncologists on a patient-specific basis. whole-genome sequencing. The fragments will have a
With the increase in novel treatment strategies, gene mixture of 5′ and 3′ overhangs, some phosphorylated. To
variants and combinations of gene variants previously facilitate ligation to synthetic adaptors, single-stranded
not considered actionable can become so. Whole-exome fragment ends are removed or filled in with nuclease or
sequencing is a method of gene discovery. This more polymerase treatment. The 5′ ends are phosphorylated.
challenging approach with regard to interpretation has The 3′ ends can be adenylated to further enable ligation
proven beneficial in cases of suspected inherited gene to adapters with T overhangs (Fig. 9.16).
variants.27,28 Initially, beyond the scope of clinical anal- Adapters are synthetic short dsDNA pieces carrying
ysis, whole-exome and even whole-genome sequencing sequences complementary to a single primer pair. The
have been increasingly incorporated in special cases. adaptors may also contain short sequences that will iden-
For routine clinical laboratory work, however, small- tify the sample (indexing or bar coding; Fig. 9.17). This
to medium-size 15- to 500-gene panels account for the allows analysis of multiple samples in the same reaction
majority of sequencing procedures. as the sequencing software will put together sequences
Sample genomic DNA

5'PO
OP3'
Fragmentation
OP
PO
End repair and

addition of adapters
Indexing
FIGURE 9.17 After fragmentation, end repair, and adapter ligation, bar codes or indexing may be performed by PCR amplification
with tailed primers, or alternatively, the index sequences may be included in the adapters. Indexes are patient-specific so that mul-
tiple patient DNA can be sequenced in the same reaction and separated by their bar codes or indices after the sequencing is
completed.
from fragments with the same bar code. Small genomes The regions to be sequenced are enriched by probe
such as those of microbes or plasmids can be simulta- hybridization or by amplification with region-specific
neously fragmented and ligated to sequencing adapters primers.29
in a single reaction tube. Reagent sets are commercially Probes are biotinylated oligonucleotides comple-
available for library preparation. mentary to specific gene regions (Fig. 9.18). Targeted
fragments to be sequenced are selected by hybridization
with the biotinylated probe and captured with strepta-
Targeted Libraries
vidin-coated beads. The captured regions are ligated to
Routine clinical sequencing of human DNA does not adapters carrying primer-binding sites (or amplified with
include the entire genome. Gene panels ranging from a primer-binding sites included with short oligo probes) so
few genes to whole exomes (all protein-coding regions) that all reactions can proceed under the same amplifi-
are employed, depending on the purpose for sequencing. cation conditions in a single PCR reaction. Probe-based
Sample genomic DNA

5'PO
OP3'
Fragmentation, denaturation
Selection and capture

(Biotin)
(Biotin)
Elution and amplification

Addition of bar codes
FIGURE 9.18 Targeted library preparation for NGS using probe enrichment. Fragmented DNA is denatured and hybridized to
region-specific biotinylated probes. The probes are bead-captured, and the hybridized regions are amplified for sequencing. Probes
may be short oligomers that can be extended across the region to be sequenced. The selected regions can then be amplified with
tailed primers to add bar codes and sequencing primer-binding sites.
enrichment has the advantage of capturing sequences design is important, however, because sequence vari-
surrounding the region of interest and providing infor- ations in the primer-binding sites may lower the effi-
mation from neighboring sequences. The presence of ciency of or even prevent amplification of particular
surrounding regions should be balanced because too fragments. Loss of library fragments from the sequenced
much additional sequencing will affect the accurate regions, referred to as allele dropout, will cause inaccu-
sequencing of the targeted regions. The balance will rate assessment of variant allele frequencies. Primers can
depend on the average length of the DNA fragments. be designed to produce overlapping sequences to cover
Amplicon-based targeted libraries are selected by less optimal regions. Paired-end or mate-pair primers
multiplex PCR with gene-specific primers tailed with produce coupled sequence fragments separated by 30 to
binding sites for a secondary primer set (Fig. 9.19). 50 kb. By overlapping these reads, large variations not
After amplification, the secondary primers are tailed detectable in a few hundred base pairs such as transloca-
with index sequences that will identify (bar code or tions can be detected.
index) fragments from multiple samples in the same Both primer- and probe-based selections are affected
sequencing reaction and adapter sequences complemen- by GC-rich sequencing targets. Secondary structure
tary to immobilized oligonucleotides anchored in the lowers the binding of primers and probes. GC-rich
sequencing platform. These steps may be combined by sequences also “clamp” primers in amplicon-based
tailing the initial multiplex PCR primers with the index enrichment, lowering PCR efficiency. AT-rich regions
and adapter sequences. Amplicon-based panel selection may also be subject to poor hybridization, leading to
has the advantage of versatility and ease of use. Primer loss of sequencing template fragments.
Target-specific primers
Index
Indexing
Adapter
Sequencing template
FIGURE 9.19 Targeted library preparation for NGS using amplification enrichment. Fragmented genomic DNA is end repaired
and amplified with region-specific primers carrying binding sites for a single set of primers used in a second amplification. The
second primer set has patient-specific index (bar-code) sequences.
(Fig. 9.21). This reaction occurs hundreds of thousands

Sequencing Platforms
of times, producing sequence information from millions
After the introduction of NGS as a pyrosequencing of sequencing panel library fragments.
technology, a variety of methods were developed for In reversible dye terminator sequencing, captured
this purpose. The two most frequently used methods in or amplified fragments are hybridized to immobilized
clinical applications are ion-conductance and reversible primers on a solid surface (flow cell). The fragments
dye terminator sequencing (Fig. 9.20). Both involve hybridize to the immobilized primers and are amplified
sequencing by synthesis and can be compared, chemi- by branch PCR into collections of products or polonies
cally, to pyrosequencing and Sanger sequencing. (Fig. 9.20B). Proper concentration (6 to 20 pMol) of the
For ion-conductance sequencing, indexed libraries library DNA introduced to the flow cell will ensure that
(gene panels) are amplified using primers immobilized the polonies are evenly spaced on the flow cell. The pol-
on microparticles (beads) in an aqueous oil emulsion onies are sequenced in place by the sequential addition
using adapters on the library fragments complementary of fluorescently labeled nucleotides. If a nucleotide is
to the immobilized primers (Fig. 9.20A). The beads car- complementary to the template next to the primer, DNA
rying the amplicons (sequence templates) are placed on polymerase will extend the primer (form a phosphodi-
a solid surface (gene chip). The captured fragments are ester bond). As in Sanger sequencing, each nucleotide is
subjected to the addition of nucleotides in a predeter- labeled with a specific color of fluor. An image is taken
mined order. If the nucleotide is complementary to the of the flow cell after each nucleotide addition (cycle),
sequencing template, DNA polymerase will catalyze recording the presence of each added nucleotide color
the formation of a phosphodiester bond. A hydrogen and location. After imaging, the fluorescent dyes are
ion is released upon formation of the phosphodiester removed, and the next nucleotide is added (Fig. 9.22).
bond. The hydrogen ion will lower the pH of the reac- Simultaneously, hundreds of thousands of polonies are
tion by a specific amount recorded by the sequencer sequenced in this way.
Beads placed on chip for

ion-based sequencing
Amplification on
beads by ePCR
A
B
Colonies formed for
reversible dye terminator
sequencing
FIGURE 9.20 (A) Library amplification for ion-conductance sequencing is performed in emulsion PCR. The bar-coded libraries
prepared are amplified from primer-binding sites complementary to bead-immobilized primers. At the end of the ePCR reaction,
the emulsion is broken and applied to a solid surface (chip) for sequencing. (B) For reversible dye terminator sequencing, the panel
is amplified by bridge PCR through primer-binding sites complementary to primers immobilized on the flow cell. Amplification in
place on the solid surface produces batches or polonies of sequencing templates distributed evenly across the flow cell.
Pyrophosphate
Nucleotide H+
pH
Template
DNA pol forms phosphodiester bond pH drops when a complementary

base is added
FIGURE 9.21 In ion-conductance sequencing, when the nucleotide added to the reaction is complementary to the template, it is
joined to the growing chain by DNA polymerase, releasing a hydrogen ion and drop in pH identifying that nucleotide. Sequencing
software converts pH changes to the nucleotide sequence.
Both sequencing platforms are accurate and efficient, Other sequencing platforms such as sequencing
with comparable performance.30 Proper controls include by ligation31 and nanopore sequencing32 are used in
a no-template sequencing control and a reference research applications. Sequencing by ligation uses a pool
sequence control. Sequence runs take from 2.5 hours to of labeled oligonucleotide DNA ligase to identify the
2 days, depending on the platform and the size of the template sequence through the known probe sequences
library being sequenced. (Fig. 9.23). Nanopore sequencing has the advantage of
Labeled Imaging
nucleotides
DNA pol forms phosphodiester bond
Dye removed
Complementary nucleotides are

distinguished by fluorescent color
FIGURE 9.22 In reversible dye terminator sequencing, labeled nucleotides are applied to the flow cell and incorporated into
growing chains by DNA polymerase at each polony location. Images are taken after rounds of fluorescent nucleotide addition; the
color at each polony location indicates the next nucleotide in that sequence. Once the image is taken, the fluorescent labels are
removed. Following this, another round of nucleotides is introduced.
Ligation
Detection
Cleavage
FIGURE 9.23 Sequencing by ligation uses short fluorescently labeled oligomers that hybridize in short increments if they are
complementary to the DNA template. The template DNA anchored to a glass slide is flooded with fluorescent-labeled oligonucle-
otides. If the oligo is complementary to the template, it is ligated, and then two bases are detected at a time. The oligonucleotide
is cleaved, followed by the next round of ligation. Each time, two new nucleotides are detected.
Current
Sequence A A C T C G T
Single-molecule sequencing (no amplification)
FIGURE 9.24 Long-read single-molecule sequencing uses protein ion channels through which one strand of each double-stranded
DNA template is drawn. Each nucleotide passing through the pore changes the current in a characteristic way. This sequencing is
rapid and does not require reassembly or short fragments for the final sequence.
not requiring fragmentation and amplification of the The next step is variant identification based on com-
template DNA. One strand of long dsDNA molecules parison with the reference sequence. There are different
(up to 1 Mb) is drawn through protein pores. Each types of variants, including single-nucleotide variants
nucleotide is identified by a disruption in current as it (SNVs), small insertion and/or deletion of nucleotides
passes through the pore (Fig. 9.24). This technology can (indels), rearrangement of sequences (e.g., transloca-
also be used for direct RNA sequencing. Development tions), and copy-number variants (CNV; amplification
of different technologies and improvement of existing or deletion of larger regions). Each of these types is
technologies are actively occurring to further facilitate handled differently by comparison software. Consti-
and widen the use of NGS. tutional (genetically inherited) SNVs are identified in
some programs based on a specific range of expected
allele frequencies (variant allele/reference allele) for
Sequence Quality
homozygosity or heterozygosity. Indels (up to 20 bp)
Instrument collection and sequencing software will batch can be identified by realignment, that is, multiple align-
the sequences for each sample, based on the bar codes, ments (offset by one or more bases) that minimize base
and identify the nucleotide order in the process of base mismatches. Indels and even larger rearrangements can
calling. Each base is assessed for quality of imaging (or be detected by overlapping reads of paired end-primed
conductance detection) and given a Phred score. Just as sequences or by points of sequence diversions from
in Sanger sequencing, a Phred score of 2 to 3 (100- to 5′ and 3′ end reads (split-read analysis). Translocation
1,000-fold certainty of a correct call) is acceptable. breakpoints are often within introns or repetitive DNA
Each sequence is then compared to a reference sequences, or they contain overlaying sequence changes
sequence through read alignment. Reference sequences at the breakpoint, posing further challenges for variant
are considered “normal” in that there are no known identification. Optimal variant detection requires the use
significant variants; however, there is no real “normal” of the appropriate library primer design and software.
sequence, especially for human DNA. Variations from Once aligned, sequence variations from the reference
the reference may be the majority allele in the popu- (variants) are arranged in a variant call file (VCF). The
lation, with the reference sequence carrying the minor VCF is a textual file that may be archived for further ref-
allele. For human genome sequencing, reference genome erence. Every variant is not of biological or clinical con-
hg19 was frequently used and reference genomes are sequence. Some variants are synonymous or silent with
updated periodically.33 Reference sequences are free of regard to protein sequences. Others are common poly-
known disease-related alleles, at least those found in the morphisms found in the population. Therefore, annota-
targeted panels. tion are performed to identify critical variants.
along with the type of variant (SNP, insertion, deletion,

TABLE 9.3 Annotation of Sequence Variants or complex). The variant is then subjected to filtering.
SNPs are compared to previously reported variants iden-
Data Description tified as human genome polymorphisms with the SNP
Location of Chromosome number, genomic rs identification number. Variants may be categorized as
variant coordinate (hg19 build) genetic or somatic in origin and, if genetic, as homo-
zygous or heterozygous with the reference allele. Some
Variant change Reference allele, alternate allele detected variants are naturally occurring polymorphisms in partic-
in sample
ular populations. Data from the 1000 Genomes Project
Genetic state Heterozygous, homozygous alternate from major ethnic populations can be used to determine
if a sequence variant is naturally present.
Quality of Quality/confidence score, sequencing
variant call depth at variant site (number of reads of
For gene panels and exome sequencing, variants will
variant and reference), probability of the likely be found in gene-coding regions and adjacent
reference or variant reads being balanced intronic sequences, although intergenic areas may also
between + and – strands be covered. The particular gene affected and the location
of the variant in exon, intron, or intergenic sequences
Allele burden The fraction of reads supporting
the alternate allele (expect germline are noted. For variants found in introns, any effects of
heterozygous alleles to be close to 0.5) splicing are assessed. Variant effects on protein can also
be estimated using algorithms such as PolyPhen and
Variant type The type of allele, either SNP, MNP, ins,
SIFT.34 Silent variants will not change the amino acid
del, or complex
sequence, but codon usage may have an effect on trans-
Genomic Exon, intron, intergenic, other lation efficiency. Conservative amino acid substitutions
position or those late in the protein sequence have less effect on
Comparison dbSNP ID, 1000 Genomes Project protein function than nonconservative mutations located
to known frequency with ethnicity, other disease- early in the protein sequence. Algorithms provide scores
variants specific databases and information to indicate the degree of damage to protein structure or
function caused by the sequence variant. The dbNSFP
Gene/coding Annotated gene at the variant site, amino
effects acid change, effect on protein sequence database is a collection of in silico detected nonsynony-
by the variant, algorithm scores for mous variants.
predicting damaging mutations Variants that remain after filtering may be annotated
by searching in disease-specific databases, such as the
Cancer Genome Atlas (TCGA), the Catalogue of Somatic
Mutations in Cancer (COSMIC), My Cancer Genome,
the Leiden Open (source) Variation Database (LOVD),
Filtering and Annotation
and the Human Gene Mutation Database (HGMD).
There are several components of annotation (Table 9.3). These databases and others contain population and clin-
The confidence in the variant call is determined by ical data associated with previously observed variants.
sequence quality and coverage. Coverage is the number The information from these databases can assist with the
of times the region containing the variant is sequenced interpretation of the clinical effect of a variant. There
from independent fragments (read depth). Coverage is are ongoing efforts to consolidate variant/disease data to
critical for confident detection of variants that are of low ever larger and more comprehensive collections. Final
frequency in the sample such as somatic mutations in reports of variants may contain information from data-
heterogeneous tumor tissue. Coverage of at least 500× bases, including effects on therapeutic treatments, espe-
(total of forward and reverse sequences) is recommended cially targeted therapies, clinical trials, and prognosis.
for detection of somatic variants. The clinical significance of a variant may differ with the
The chromosomal and sequence location of the variant heterogeneity of disease states as well as patient charac-
in context with the reference sequence is identified, teristics and demographics (e.g., age or gender).
to communicate consensus sequences and for computer

Advanced Concepts input of polymorphic sequence data.
In addition to the interpretation of sequence variants,
Based on professional surveys and literature sequence information is also used in epidemiology, to spe-
reviews, a multidisciplinary group has proposed a ciate organisms or to find homologies within or between
system to categorize somatic variants in cancer.35 species. These applications involve database searches
It defines four tiers of variants as determined with comparisons of large regions of DNA. The Basic
from cancer variant databases: tier I, variants with Local Alignment Search Tool (BLAST) is a system for
strong clinical significance; tier II, variants with homology searches. BLAST searches GenBank, a large
potential clinical significance; tier III, variants of database maintained by the National Center for Biotech-
unknown significance (VUS); and tier IV, variants nology Information (NCBI). Searches can be made of
likely to be benign. nucleic acid and amino acid sequences. Searches are
performed by selecting a nucleotide or protein search
and entering a sequence (query). Limits and parameters
on the search can be added, such as the type of organ-
isms to search (e.g., human, mouse, or other), exclusions
BIOINFORMATICS and limits of organism or sample type, and the program.
The program can optimize for highly similar sequence
Information technology has had to encompass the vast matches (megablast) or imperfect matches. Because
amount of data arising from the growing numbers of sequences are directly submitted by researchers, there
sequence discovery methods, especially direct sequenc- may be differences in the entered sequences due to the
ing and array technology. This deluge of information source of the sequenced material, the sequencing method,
requires careful submission, storage, organization, and or the quality of the sequence. Selecting less-than-perfect
indexing of large amounts of data into databases such matches will also allow cross-species matches of phy-
as those used in clinical sequencing analysis. Bioinfor- logenically conserved sequences, which can lead to the
matics is the merger of biology with information tech- identification of important protein domains or clues to
nology. Part of the practice in this field is biological protein function.
analysis in silico, that is, by computer. Bioinformatics The search will generate a number of matches or hits,
dedicated specifically to handling sequence information with a diagram showing the alignments of the matching
is a form of computational biology. A list of some of sequences and a color code indicating the best matches.
the terms used in bioinformatics is shown in Table 9.4. Another section of the search results in E-values. The
The handling of the mountains of data being generated E-value (Expect value) describes the number of matches
requires continual renewal of stored data, and a number to the query by chance when searching a database of
of database programs are available for this purpose.36,37 a particular size. It decreases exponentially with the
Standard expression of sequence data is important quality of the match. Very low E-values (e.g., 10–12)
for the clear communication and organized storage of would be associated with a perfect match for a given
sequence data. In some cases, such as in heterozygous query sequence. Further information, including the
mutations, there may be more than one base or mixed matched gene name and its organism, the source of the
bases at the same position in the sequence. Polymorphic matched sequence and the location within that sequence,
or heterozygous sequences are written as consensus comparison of base to base or amino acid to amino
sequences, or a family of sequences, with proportional acid, and plus or minus strand of the matched nucleo-
representation of the polymorphic bases. The Inter- tide sequence, are accessed by selecting the sequence or
national Union of Pure and Applied Chemistry and the color-coded bar in the diagram. The original submit-
the International Union of Biochemistry and Molec- ted sequence can be accessed by selecting the sequence
ular Biology (IUB) have assigned a universal nomen- name.
clature for mixed, degenerate, or wobble bases (Table In addition to the identification of new sequences,
9.5). The base designations in the IUB code are used queries such as these are also useful for test and primer
TABLE 9.4 Bioinformatics Terminology
Term Definition
Identity The extent to which two sequences are the same
Alignment Lining up two or more sequences to search for the maximal regions of identity in order to
assess the extent of biological relatedness or homology
Local alignment Alignment of some portion of two sequences
Multiple sequence alignment Alignment of three or more sequences arranged with gaps so that common residues are
aligned together
Optimal alignment The alignment of two sequences with the best degree of identity
Conservation Specific sequence changes (usually protein sequence) that maintain the properties of the
original sequence
Similarity The relatedness of sequences, the percent identity or conservation
Algorithm A fixed set of commands in a computer program
Domain A discreet portion of a protein or DNA sequence
Motif A highly conserved short region in protein domains
Gap A space introduced in alignment to compensate for insertions or deletions in one of the
sequences being compared
Homology Similarity attributed to descent from a common ancestor
Orthology Homology in different species due to a common ancestral gene
Paralogy Homology within the same species resulting from gene duplication
Query The sequence presented for comparison with all other sequences in a selected database
Annotation Description of functional structures, such as introns or exons in DNA or secondary structure or
functional regions to protein sequences
Interface The point of meeting between a computer and an external entity, such as an operator, a
peripheral device, or a communications medium
GenBank The genetic sequence database sponsored by the National Institutes of Health
PubMed Search service sponsored by the National Library of Medicine that provides access to literature
citations in Medline and related databases
SwissProt Protein database sponsored by the Medical Research Council (United Kingdom)
design. Whenever a new primer or probe sequence is probes with multiple potential binding sites will produce
chosen, it is useful to query the primer or probe sequence mis-primes and off-target products.
to confirm that it belongs to the correct species and is not Bioinformatics includes handling and updating
duplicated in multiple places in a genome. Primers and of information for software tools and databases. The
of the arrangement of its nucleotides have attracted great

TABLE 9.5 IUB Universal Nomenclature interest. Gradually, this information began to accumu-
for Mixed Bases late, first regarding simple microorganisms and then par-
tially in lower and higher eukaryotes. The deciphering
Symbol Bases Mnemonic of the human genome was a benchmark in the ongoing
A Adenine Adenine discovery of the molecular basis for disease and the
groundwork of molecular diagnostics. In the process of
C Cytosine Cytosine solving the human DNA sequence, genomes of a variety
G Guanine Guanine
of clinically important organisms were deciphered,
advancing typing and predicting infectious disease treat-
T Thymine Thymine ment outcomes.
The first complete genome sequence of a clinically
U Uracil Uracil
important organism was that of Epstein–Barr virus,
R A, G puRine published in 1984.38 The 170,000-bp sequence was
determined using the M13 template preparation/chain
Y C, T pYrimidine
termination manual sequencing method. In 1985 and
M A, C aMino 1986, the possibility of mapping or sequencing the
human genome was discussed at meetings at the Uni-
K G, T Keto
versity of California, Santa Cruz; Cold Spring Harbor,
S C, G Strong (3 H bonds) New York; and the Department of Energy in Santa Fe,
New Mexico. The idea was controversial because of the
W A, T Weak (2 H bonds)
risk that the $2 to $5 billion cost of the project might
H A, C, T Not G not justify the information gained, most of which would
be sequences of “junk,” or non-gene-coding DNA. Fur-
B C, G, T Not A
thermore, there was no available technology up to the
V A, C, G Not T massive task. The sequencing automation and the com-
puter power necessary to assemble the 3 billion bases
D A, G, T Not C
of the human genome into an organized sequence of
N A, C, G, T aNy 23 chromosomes had not yet been developed.
Nevertheless, several researchers, including Walter
X, ? Unknown A or C or G or T Gilbert (of Maxam–Gilbert sequencing), Robert
O, - Deletion Sinsheimer, Leroy Hood, David Baltimore, David
Botstein, Renato Dulbecco, and Charles DeLici, saw that
the project was feasible because technology was rapidly
advancing toward full automation of the process. In
accumulation of genomic and proteomic data, species 1982, Akiyoshi Wada had proposed automated sequenc-
and types of microorganisms based on sequences data, ing machinery and had gotten support from Hitachi
and variant association with disease drives the devel- Instruments. In 1987, Smith and Hood announced the
opment of high-powered, reliable computer systems for first automated DNA sequencing machine.39 Advances in
storage as well as organization. the chemistry of the sequencing procedure were accom-
panied by advances in the biology of DNA mapping,
with methods such as pulsed-field gel electrophoresis,40,41
THE HUMAN GENOME PROJECT restriction fragment length polymorphism analysis,42
and transcript identification. Methods were developed to
From the first description of its double-helical structure clone large (500-kbp) DNA fragments in artificial chro-
in 1953 to the creation of the first recombinant molecule mosomes, providing long contiguous sequencing tem-
in the laboratory in 1972, DNA and the chemical nature plates.43 Finally, application of capillary electrophoresis
Genomic Research (TIGR). Venter ’s group completed

TABLE 9.6 Model Organisms Sequenced the first sequence of a free-living organism (Haemophilus
During the Human Genome Project influenzae)49 and the sequence of the smallest free-living
organism (Mycoplasma genitalium).50 Venter established
Estimated a new company named Celera and proposed to complete
Genome Number
Organism Size (Mb) of Genes the human genome sequence in 3 years for $300 million,
faster and cheaper than the NIH project. Meanwhile,
Epstein–Barr virus 0.17 80 Watson had resigned as head of the NIH project and was
replaced by Francis Collins. In response, the Wellcome
Mycoplasma genitalium 0.58 470
Trust doubled its support of the NIH project. The NIH
Haemophilus influenzae 1.8 1,740 moved its completion date from 2005 to 2003, with a
working draft to be completed by 2001. Thus began a
Escherichia coli K-12 4.6 4,377
competitive effort on two fronts to sequence the human
E. coli O157 5.4 5,416 genome.
The two projects approached the sequencing differ-
Saccharomyces cerevisiae 12.5 5,770
ently (Fig. 9.25). The NIH method (hierarchical shotgun
Drosophila melanogaster 180 13,000 sequencing) was to start with sequences of known
regions in the genome and “walk” further away into the
Caenorhabditis elegans 97 19,000 chromosomes, always aware of where the newly gen-
Arabidopsis thaliana 90 25,000 erated sequences belonged in the human genome map.
Venter and the researchers working with Celera—Gene
Meyers, Jane Rogers, Robert Millman, John Sulston,
and Todd Taylor—had a different idea. Their approach
(whole-genome shotgun sequencing) was to start with
to DNA resolution44–46 made the sequencing procedure 10 equivalents of the human genome cut into small frag-
even more rapid and cost-efficient. ments and randomly sequence the lot. Then, powerful
With these developments in technology, the Human computers would find overlapping sequences and use
Genome Project was endorsed by the National Research those to assemble the billions of bases of sequence into
Council. The National Institutes of Health (NIH) estab- their proper chromosomal locations.
lished the Office of Human Genome Research with Initially, the Celera approach was met with skepti-
James Watson as its head. Over the next 5 years, meet- cism. The human genome contains large amounts of
ings on policy, ethics, and the cost of the project resulted repeated sequences, some of which are very difficult
in a plan to complete 20 Mb of sequence of model organ- to sequence and even more difficult to map properly.
isms by 2005 (Table 9.6). To organize and compare the A random sequencing method would repeatedly cover
growing amount of sequence data, the BLAST and Gene areas of the genome that are more easily sequenced
Recognition and Assembly Internet Link (GRAIL) algo- and miss more difficult regions. Moreover, assembly of
rithms were introduced in 1990.47,48 the whole sequence from scratch with no chromosomal
For the human sequence, the decision was made to landmarks would take a prohibitive amount of computer
use a composite template from multiple individuals power. Nonetheless, Celera began to make headway
rather than a single genome from one donor. Human (some alleged with the help of the publicly published
DNA was donated by 100 anonymous volunteers; only sequences from the NIH), and eventually, the NIH
10 of these genomes were sequenced. Not even the vol- project modified its approach to include both methods.
unteers knew if their DNA was used for the project. To Over the next months, some efforts were made toward
ensure accurate and high-quality sequencing, all regions combining the two projects, but these efforts broke down
were sequenced 5 to 10 times. over disagreements over database policy and release of
A second project started with the same goal. In completed sequences. The result of the competition was
1992, Craig Venter left the NIH to start the Institute for that the rough draft of the sequence was completed by
Hierarchical Shotgun Sequencing Whole-Genome Shotgun Sequencing
Whole genome
Known regions
of individual
chromosomes
Random reads
Assembly
Anchoring
Genome assembly
FIGURE 9.25 Comparison of two approaches for sequencing of the human genome. The hierarchical shotgun approach taken by
the NIH (left) was to sequence from known regions so that new sequences could easily be located in the genome. The Celera
whole-genome shotgun approach (right) was to sequence random fragments from the entire genome and then assemble the com-
plete sequence with computers.
both projects earlier than either group had proposed, in GC base pairs (25%). A most surprising discovery was
June 2000. A joint announcement was made, and both that the number of genes, estimated to be from 20,000
groups published their versions of the genome, the NIH to 30,000, was much lower than expected. The average
version in the journal Nature51 and the Celera version in size of a human gene is 27 kbp. Chromosome 19 is the
the journal Science.52 most gene-rich per unit length (23 genes/Mbp). Chro-
The sequence completed in 2000 was a rough draft mosomes 13 and Y have the fewest genes per base pair
of the genome; that is, there were still areas of missing (5 genes/Mbp). Only about 2% of the sequences code for
sequence and sequences yet to be placed. Only chro- genes. Between 30% and 40% of the genome consists
mosomes 21 and 22, the smallest of the chromosomes, of repeat sequences. There is one single-base difference
had been fully completed. In the ensuing years, the fin- between two random individuals found approximately
ished sequences of each chromosome have been released every 1,000 bases along the human DNA sequence.
(Table 9.7). More detailed information, databases, references, and
Remaining errors, gaps, and complex gene rearrange- updated information are available at http://www.ncbi
ments will take years to resolve.53 Detailed analysis of .nlm.nih.gov/.
an individual genome will require sequencing of both The promise of the Human Genome Project for
homologs of each chromosome.54 Even with the rough molecular diagnostics can be appreciated with the
draft, interesting characteristics of the human genome example of the discovery of the gene involved in cystic
were revealed. The size of the entire genome is 2.91 Gbp fibrosis. Seven years of work were required for discov-
(2.91 billion bp). The genome was initially calculated ery of this gene. With proper mapping information, a
as 54% AT and 38% GC, with 8% of the bases still gene for any disease can now be found by computer,
to be determined. Chromosome 2 is the most GC-rich already sequenced, in a matter of minutes. Of course,
chromosome (66%), and chromosome X has the fewest all genetic diseases are not due to the malfunction of
almost impossible. Ten years after the announcement of

TABLE 9.7 Completed Chromosomes the completion of the Human Genome Project, almost
200 human genomes had been sequenced. Although the
Chromosome Completion Date information gathered from the sequencing effort had not
21 December 1999 yielded the benefits to human health expected at the start
of the project, it increased the appreciation of the vast
22 May 2000 complexity of genes and their regulation.55
20 December 2001
14 January 2003 Variant Associations With Phenotype

Y June 2003 The Human Haplotype Mapping Project
7 July 2003 As the Human Genome Project moved toward com-
pletion, another project was launched to further define
6 October 2003
the relationship between gene sequence and disease.
13 March 2004 This was the Human Haplotype Mapping, or HapMap,
Project.56 The goal of the project was to find blocks of
19 March 2004
sequences that are inherited together, marking particu-
9 May 2004 lar traits and possibly disease-associated genetic lesions.
The haplotype approach would reduce the number of
9 May 2004
polymorphisms required to examine the entire col-
5 September 2004 lection of genome/phenotype associations from the
10 million polymorphisms that exist to roughly
16 December 2004
500,000 haplotypes. The HapMap Project revealed more
18 January 2005 than 1,000 disease-associated regions of the genome,
covering commonly occurring conditions such as coro-
X March 2005
nary artery disease and diabetes. With advances in tech-
2 April 2005 nology, and the ability to generate sequence information,
however, HapMap data has mostly been supplanted by
4 April 2005 higher-throughput data-gathering methods. As a result,
8 January 2006 the NCBI has planned to retire the HapMap project site
because other resources, such as the 1000 Genomes
11 March 2006 Project, have become more comprehensive references
15 March 2006 for population genomics.
12 March 2006
The 1000 Genomes Project
17 April 2006
The 1000 Genomes Project provides a resource of struc-
3 April 2006 tural variants in different populations.57 The project has
1 May 2006 reconstructed the genomes of over 2,504 individuals
from 26 populations by whole-genome sequencing, deep
exome sequencing, and dense microarray genotyping
a single gene. In fact, most diseases and normal states in laboratories in the United States, United Kingdom,
are driven by a combination of genes as well as by China, and Germany. Over 88 million variants (84.7
environmental influences. Without the rich information million SNPs, 3.6 million short insertions/deletions, and
afforded by the sequence of the human genome, iden- 60,000 structural variants) were verified. The resulting
tification of these multicomponent diseases would be database includes more than 99% of single-nucleotide
variants with a frequency of greater than 1%. Data from 3. A dideoxy sequencing electropherogram displays
the 1000 Genomes Project is a component of NGS bright (high, wide) peaks of fluorescence,
variant assessment, providing more patient-specific obliterating some of the sequencing peaks. What
interpretation of the clinical significance of variants. All is the most likely cause of this observation? How
variants from the 1000 Genomes Project are submitted might it be corrected?
to archives such as dbSNP.
The majority of HapMap SNPs are found in the 4. In a manual sequencing reaction, the sequencing
1000 Genomes Project.58 Sites from HapMap that aren’t ladder on the polyacrylamide gel is very bright
found by the 1000 Genomes Project may be false dis- and readable at the bottom of the gel, but the
coveries by HapMap, the latter being based on microar- larger (slower-migrating) fragments higher up are
ray technology. Thus, there are a lot of SNPs from NGS very faint. What is the most likely cause of this
projects that are not reported in HapMap. observation? How might it be corrected?
The technology developed as part of the Human
Genome Project made sequencing a routine method in 5. In an analysis of the TP53 gene for mutations,
the clinical laboratory. Small, cost-effective sequenc- the following sequences were produced. For each
ers are available for rapid sequencing. In the clini- sequence, write the expected sequence of the
cal laboratory, sequencing is actually resequencing, opposite strand that would confirm the presence of
or repeated analysis of the same sequence region, to the mutations detected.
detect mutations or to type microorganisms, making
the task even more routine. The technology continued 5′TATCTGTTCACTTGTGCCCT3′ (Normal)
to develop, reducing the cost and labor of sequencing 5′TATCTGTTCATTTGTGCCCT3′ (Homozygous
to detect multicomponent diseases or to predict predis- substitution)
position to disease. Massive parallel or next-generation 5′TATCTGT(T/G)CACTTGTGCCCT3′
sequencing has supplemented and/or replaced Sanger (Heterozygous substitution)
sequencing in many clinical laboratories, and even this 5′TATCTGTT(C/A)(A/C)(C/T)T(T/G)(G/T)
technology has evolved into lower-cost, user-friendly (T/G) (G/C)CC(C/T) . . . 3′ (Heterozygous
protocols. Accurate and comprehensive sequence anal- deletion)
ysis is one of the most promising areas of molecular
diagnostics. 6. A sequence, TTGCTGCGCTAAA, may be
methylated at one or more of the cytosine residues.
After bisulfite sequencing, the following results are
obtained:
STUDY QUESTIONS Bisulfite treated: TTGUTGCGUTAAA

Write the sequence showing the methylated
cytosines as CMe.
1. Read 5′ to 3′ the first 15 bases of the sequence in
the gel on the right in Figure 9.7 (p. 229). 7. In a pyrosequencing readout, the graph shows
peaks of luminescence corresponding to the
2. After an automated dye primer sequencing run, the addition of the following nucleotides:
electropherogram displays consecutive peaks of the
following colors: dT peak, dC peak (double height), dT peak,
red, red, black, green, green, blue, black, red, dA peak
green, black, blue, blue, blue What is the sequence?
If the computer software displays the fluors from
ddATP as green, ddCTP as blue, ddGTP as black, 8. Why is it necessary to add adenosine residues
and ddTTP as red, what is the sequence of the in vitro to ribosomal RNA before capture for
region given? sequencing?
9. Which of the following is next-generation 12. Shiraishi M, Hayatsu H. High-speed conversion of cytosine to
sequencing? uracil in bisulfite genomic sequencing analysis of DNA methyla-
tion. DNA Research 2004;11:409–415.
a. Maxam–Gilbert 13. Weller M, Tabatabai G, Kästner B, Felsberg J, Steinbach JP,
b. Tiled microarray Wick A, Schnell O, Hau P, Herrlinger U, Sabel MC, Wirsching
HG, Ketter R, Bähr O, Platten M, Tonn JC, Schlegel U, Marosi
c. Dideoxynucleotide chain terminator C, Goldbrunner R, Stupp R, Homicsko K, Pichler J, Nikkhah G,
sequencing Meixensberger J, Vajkoczy P, Kollias S, Hüsing J, Reifenberger G,
d. Reversible dye terminator sequencing Wick W; DIRECTOR Study Group. MGMT promoter methylation
is a strong prognostic biomarker for benefit from dose-intensified
10. Which of the following projects would require temozolomide rechallenge in progressive glioblastoma: the
DIRECTOR trial. Clinical Cancer Research 2015;21:2057–2064.
next-generation sequencing? 14. Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent
a. Mapping a mutation in the hemochromatosis MJ, Wick W, Hegi ME. MGMT promoter methylation in malig-
nant gliomas: ready for personalized medicine? Nature Reviews
gene Neurology 2010;6:39–51.
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Section III
Techniques in the
Clinical Laboratory
259
Chapter 10
DNA Polymorphisms
and Human Identification
Outline SINGLE-NUCLEOTIDE POLYMORPHISMS

The Human Haplotype Mapping (HapMap) Project
TYPES OF POLYMORPHISMS MITOCHONDRIAL DNA POLYMORPHISMS
RFLP TYPING OTHER IDENTIFICATION METHODS
Genetic Mapping With RFLPs Protein-Based Identification
RFLP and Parentage Testing Epigenetic Profiles
Human Identification Using RFLPs
DNA Fingerprinting With RFLP
STR TYPING BY PCR
STR Analysis Objectives
STR Nomenclature
Gender Identification 10.1 Compare and contrast different types of
Analysis of Test Results polymorphisms.
Genotyping 10.2 Define restriction fragment length polymorphism
Matching of Profiles (RFLP), and discuss how RFLPs are used in
Allelic Frequencies in Paternity Testing genetic mapping, parentage testing, and human
Sibling Tests identification.
Y-STR 10.3 Describe short tandem repeat (STR) structure
Matching With Y-STRs and nomenclature.
LINKAGE ANALYSIS 10.4 Describe the use of STR in parentage testing and
BONE MARROW ENGRAFTMENT TESTING USING the amelogenin locus for gender identification.
DNA POLYMORPHISMS 10.5 Explain matching probabilities and the
PSTR Testing contribution of allele frequencies to the certainty
Post-Transplant Engraftment Testing of matching.
QUALITY ASSURANCE OF TISSUE SECTIONS USING STR 10.6 Describe the use of Y-STR in forensic and lineage
studies.
260
Chapter 10 • DNA Polymorphisms and Human Identification 261
10.7 Use STR for linkage analysis. are more than 500,000 of these LINE-1 (L1) elements,
10.8 Give examples of the use of STR for bone making up more than 15% of the human genome. There
marrow engraftment monitoring. are even more short interspersed nucleotide elements
10.9 Show how STR may be used for quality (SINEs) scattered over the genome. SINEs, 0.3 kbp in
assurance of histological sections. size, are present in over 1,000,000 copies per genome.
10.10 Define single-nucleotide polymorphism (SNP), and SINEs include Alu elements, named for harboring rec-
explain the potential use of SNPs in disease gene ognition sites for the AluI restriction enzyme. There are
mapping. well in excess of 1 million Alu elements, accounting for
10.11 Discuss mitochondrial DNA typing. almost 11% of the human genome.1 The majority of tran-
10.12 Identify a protein profile from a reference scribed genes contain Alu elements in their introns. Alu
database. elements have cryptic splice and polyadenylation sites,
10.13 Predict the effect of aging on epigenetic (DNA which can become activated through the accumulation
methylation) profiles. of mutations and lead to alternative splicing of RNAs or
premature termination of translation. LINEs and SINEs
are also known as mobile elements or transposable ele-
Polymorphisms are variations of DNA sequences that ments. They are copied and spread by recombination
are shared by a certain percentage of a population. These and reverse transcription and may be responsible for
sequences range from a single base pair to thousands of the formation of pseudogenes (intronless, nonfunctional
base pairs. copies of active genes) throughout the human genome.
Shorter blocks of repeated sequences also undergo
expansion or shrinkage through generations. Examples
TYPES OF POLYMORPHISMS of the latter are short tandem repeats (STRs) and vari-
able-number tandem repeats (VNTRs).
The probability of polymorphic DNA in humans is great SNPs, larger sequence variants, and tandem repeats
due to the relatively large size of the human genome, can be detected by observing changes in the restriction
98% of which does not code for genes. At the nucleotide- map of a DNA region. Analysis of restriction fragments
sequence level, it is estimated that genome sequences by Southern blot reveals restriction fragment length
differ by at least one nucleotide every 1,000 to 1,500 polymorphisms (RFLPs). Particular types of polymor-
bases. These single-nucleotide differences, or single- phisms, specifically SNPs, VNTRs, STRs, and RFLPs,
nucleotide polymorphisms (SNPs), may occur in are routinely used in the laboratory (Table 10.1).
gene-coding regions or in intergenic sequences.
Polymorphisms are more frequent in some areas of
the genome than in others. The human leukocyte antigen Histooricaal Higghlligghtts
(HLA) locus is a familiar example of a highly polymor-
phic region of human DNA. The variable nucleotide In the 1920s, scientists realized that blood type
sequences in this locus code for peptides that establish (A, B, AB, or O) is inherited and could be used
self-identity of the immune system. The extent of sim- for parentage testing. This limited testing could
ilarity or compatibility between the immune systems of only exclude a falsely alleged father. But soon
transplant recipients and potential donors can thus be after, the use of other proteins on the surface of
determined by comparing DNA sequences. Some human the red blood cell (Rh, Kell, and Duffy blood
sequence polymorphisms affect many base pairs. Large group systems) was introduced. The power of
blocks of repeated sequences may be inverted, deleted, these serological tests was only marginally better
or duplicated from one individual to another. Long than that of the ABO system. Forty years later,
interspersed nucleotide elements (LINEs) are highly the polymorphic HLAs were implemented for
repeated sequences, 6 to 8 kbp in length, that contain parentage and identity testing, coupled with the
RNA polymerase promoters and open reading frames ABO and serological testing.
related to the reverse transcriptase of retroviruses. There
262 Section III • Techniques in the Clinical Laboratory
number of fragments generated by restriction enzyme

TABLE 10.1 Types of Useful Polymorphisms digestion of DNA (Fig. 10.1). Fragment sizes vary as a
and Laboratory Methods result of changes in the nucleotide sequence in or between
the recognition sites of a restriction enzyme. Nucleotide
Detection changes may also destroy, change, or create restriction
Polymorphism Structure Method
enzyme sites, altering the number of fragments.
RFLP One or more nucleotide Southern The first step in using RFLPs is to construct a restric-
changes that affect blot tion enzyme map of the DNA region under investigation.
the size of restriction Once the restriction map is known, the number and sizes
enzyme products of the restriction fragments of a test DNA region cut
VNTR Repeats of 10–50 base Southern with restriction enzymes are compared with the number
sequences in tandem blot, PCR and sizes of fragments expected based on the restriction
map. Polymorphisms are detected by observing fragment
STR Repeats of 1–10 base PCR
sequences in tandem
numbers and sizes different from those expected from
the reference restriction map. An example of a polymor-
SNP Alterations of a single Sequencing, phism in a restriction site is shown in Figure 10.2. In a
nucleotide other theoretical linear piece of DNA, loss of the recognition
site for the enzyme (BglII in the figure) results in alter-
ation of the size and number of bands detected after gel
RFLP TYPING electrophoresis.
RFLP typing in humans required the use of the
RFLPs were the original DNA targets used for gene Southern blot technique. DNA was cut with restriction
mapping, human identification, and parentage testing. enzymes, resolved by gel electrophoresis, and blotted to
The first polymorphic RFLP was described in 1980. a membrane. Probes to specific regions of DNA contain-
RFLPs are observed as differences in the sizes and ing potential RFLPs were then hybridized to the DNA
Normal DNA Eco RI site
GTCCAGTCTAG CG AATTCG TG G CAAAG G C T

CAGGTCAGATCGCTTAAGCACCGTTTCCGA
Bal I site
Point mutations
GTCCAGTCTAG CG AAATCG TG G C CAA G G C T
CAGGTC AG ATCG CTTTAG CACCG G TTC C G A
Insertions
GTCCAGTCTAG CG AAG CG A ATTCG TG G CT C A A A G G C T FIGURE 10.1 Types of DNA sequence alter-
CAGGTCAGATCGCTTCGCTTAAGCACCGAGTTTCCGA ations that change restriction fragment lengths.
The normal sequence (top) has an EcoR1 site
Duplications (GAATTC). Single-base changes (point muta-
GTCCAGTCTAG CG AATTCG TGTAG CG A A T T C G T GG C A A A tions, second line) can destroy the EcoR1 site or
CAGGTC AG ATCG CTTAAG CACATCG CT T A A G C A CC G T T T create a new restriction site, as can insertions,
duplications, or deletions of any number of
bases (third through fifth lines). Insertions,
Fragment insertion (or deletion)
duplications, and deletions between two restric-
GTCCAGTCTAG CG AATTCG TG G CAAAA A AC A A G G C T G A A T T C tion sites change fragment size without affecting
CAGGTCAGATCGCTTAAGCACCGTTTTTTGTTCCGACTTAAG the restriction sites themselves.
Bgl II Bgl II Bgl II Bgl II

1 2 1 2
A B C A B C
Probe
+ –
AG ATCT AT ATCT
TCTAG A TA TAG A
1 2 Fragments visualized
+/+ +/– –/+ –/–
+ + B
+ – B+C
1 2 Size Number +/+ +/– –/+ –/–
– + A+B
+ + A, B, C 3 – – A+B+C
+ – A, B+C 2
– + A+B, C 2
Genotype Fragments visualized
– – A+B+C 1 I II III
I ++/+– B, B+C
II +–/–+ A+B, B+C
FIGURE 10.2 A linear piece of DNA with two polymorphic III ++/– – B, A+B+C
BglII restriction enzyme sites, designated as 1 and 2, will yield
different fragment sizes, depending on the presence of neither,
FIGURE 10.3 Using a Southern blot to probe for RFLP. With
either, or both of the restriction sites. For instance, a G to T
the same region shown in Figure 10.2, only the fragments with
mutation will change the sequence of the normal site (+) to one
complementary sequences to a probe to the B region (top) can
not recognized by the enzyme (–). The presence or absence of
be visualized. The bottom panel shows a diploid genotype
the polymorphic sites is evident from the number and size of
where homologous chromosomes carry different RFLP alleles.
the fragments after cutting the DNA with BglII (bottom right).
on the membrane to determine the size of the resulting sequences. One consequence of this genetic diversity is
bands. Figure 10.3 shows the pattern of bands result- that a single locus, that is, a gene or region of DNA,
ing from a Southern blot analysis of the RFLP in the will have several versions, or alleles. Human beings are
linear fragment from Figure 10.2. Even if the probe does diploid with two copies of every locus. In other words,
not detect all of the restriction fragments, the polymor- each person has two alleles of each locus. If these alleles
phisms can still be identified. are the same, the locus is homozygous; if the two alleles
DNA is inherited as one haploid chromosome com- are different, the locus is heterozygous.
plement from each parent. Each chromosome carries its Depending on the extent of diversity or polymorphism
polymorphisms so that the offspring inherits a combi- of a locus, any two people can share the same alleles or
nation of the parental polymorphisms. When visualized have different alleles. More closely related individuals
as fragments that hybridize to a probe of a polymorphic are likely to share more alleles than unrelated persons.
region, the band patterns represent the combination of In the examples shown in Figure 10.3, (+ +), (+ –),
RFLPs inherited from each parent. Due to recombination (– +), and (– –) describe the presence (+) or absence
and random assortment, each person has a unique set of (–) of BglII sites making up four alleles of the locus
RFLPs, half inherited maternally and half paternally. detectable by Southern blot. In the illustration, geno-
Every genotype will yield a descriptive band pattern, as types I and II both have the (+ –) allele on one chro-
shown in Figure 10.3. mosome, but genotype I has (+ +), and genotype II has
Over many generations, mutations, intra- and inter- (– +) on the other chromosome. This appears in the
chromosomal recombination, gene conversion, and other Southern blot results as one band of equal size between
genetic events have increased the diversity of DNA the two genotypes and one band that is a different size.
Two individuals can share both alleles at a single locus,

but the chances of two individuals, except for identical
twins, sharing the same alleles decrease 10-fold with Mary Claire King used RFLP to map one of the
each additional locus tested.2 genes mutated in inherited breast cancer.3,4 Fol-
More than 2,000 RFLP loci have been described in lowing extended families with high incidence of
human DNA. The uniqueness of the collection of poly- breast and ovarian cancer, she found particular
morphisms in each individual is the basis for human RFLP always present in affected family members.
identification at the DNA level. Detection of RFLP by Because the location in the genome of the RFLP
Southern blot made positive paternity testing and human was known (17q21), the BRCA1 gene was thereby
identification possible for the first time. mapped to this position on the long arm of chro-
To optimize the discriminatory capacity of RFLP mosome 17.
testing, restriction enzymes that cut human DNA fre-
quently were used for RFLP tests. RFLP protocols for
human identification in most North American laborato-
RFLP and Parentage Testing
ries used the restriction enzyme HaeIII for fragmentation
of genomic DNA. Many European laboratories used the In diploid organisms, chromosomal content is inherited,
HinfI enzyme. All of these enzymes cut DNA frequently half from each parent. This includes the DNA polymor-
enough to reveal polymorphisms in multiple locations phisms located throughout the genome. Taking advantage
throughout the genome. To regulate results from inde- of the unique combination of RFLP in each individual,
pendent laboratories, the Standard Reference Material one can infer a parent’s contribution of alleles to a child
(SRM) DNA Profiling Standard for RFLP analysis was from the combination of alleles in the child and those of
released in 1992. The SRM supplies cell pellets, genomic the other parent. The fragment sizes of an individual are
DNA, gel standards, precut DNA, electrophoresis mate- a combination of those from each parent, as illustrated
rials, molecular-weight markers, and certified values for in Figure 10.4. In a paternity test, the alleles or fragment
final analysis. These materials, currently provided by the
National Institute of Standards and Technology (NIST),
Father Mother
were designed to maintain the reproducibility of the Locus Locus
RFLP process across laboratories. A B A B
Genetic Mapping With RFLPs

Parents
Polymorphisms are inherited in a Mendelian fashion,
and the locations of many polymorphisms in the genome
are known. Therefore, polymorphisms can be used as
landmarks, or markers, in the genome to determine the Locus
A B
location of other genes. In addition to showing clear
family history or direct identification of a genetic factor,
one can confirm that a disease has a genetic component Child
by demonstrating a close genetic association or linkage
to a known marker. Formal statistical methods are used
to determine the probability that an unknown gene is
located close to a known marker in the genome. The
FIGURE 10.4 RFLP inheritance. Two different genetic
more frequently a particular polymorphism is present regions, or loci, are shown, locus A and locus B. There are
in persons with a disease phenotype, the more likely an several versions or alleles of each locus. Note that the father is
affected gene is located close to the polymorphism. This heterozygous at locus A and homozygous at locus B. The
is the basis for linkage mapping and one of the ways alleles in the child will be a combination of one allele from
genetic components of disease are identified. each parent.
sizes of the offspring and the mother are analyzed. The Human Identification Using RFLPs
remaining fragments (the ones that do not match the
mother) have to come from the father. Alleged fathers The first genetic tool used for human identification was
are identified based on the ability to provide the remain- the ABO blood group antigens. Although this type of
ing alleles (inclusion). Aside from possible mutations, a analysis could be performed in a few minutes, the dis-
difference in just one allele may exclude paternity. crimination power was low. With only four possible
A simplified RFLP paternity test is shown in groups, this method was only good for exclusion (elim-
Figure 10.5. Of the two alleged fathers shown, only one ination) of a person and was informative only in 15%
could supply the fragments not supplied by the mother. to 20% of cases. Analysis of the polymorphic HLA loci
In this example, only two loci are shown. A parentage added a higher level of discrimination, with exclusion
test requires analysis of at least eight loci. The more loci in 90% of cases. Testing both ABO and HLA did not
tested, the higher the probability of positive identifica- provide positive identification, however.
tion of the father. The initial use of DNA as an identification tool relied
on RFLP detectable by Southern blot. As shown in
Figure 10.1, RFLP can arise from a number of genetic
events, including point mutations in the restriction site,
AF 1 AF 2 Mother
mutations that create a new restriction site, and inser-
Locus Locus Locus
A B A B A B tion or deletion of repeated sequences (tandem repeats).
The insertion or deletion of nucleotides occurs fre-
quently in repeated sequences in DNA. Tandem repeats
of sequences of all sizes are present in genomic DNA
(Fig. 10.6). Repeat units can be large enough so that
loss or gain of one repeat is resolved by gel electro-
phoresis of a restriction enzyme digest. The frequent
Child cutters, HaeIII (recognition site GGCC) or HinfI (recog-
Locus nition site GANTC), generate fragments that are small
A B enough to resolve those that contain different numbers
of repeats and thereby give an informative pattern by
Southern blot.
DNA Fingerprinting With RFLP
FIGURE 10.5 Two alleged fathers (AFs) are being tested for The first human DNA profiling system was introduced by
paternity of the child whose partial RFLP profile is shown in the United Kingdom Forensic Science Service in 1985
the bottom gel. The mother ’s alleles are shown in green. One using Sir Alec Jeffreys’s Southern blot multiple-locus
AF (AF1) is excluded from paternity because he cannot supply probe (MLP)-RFLP system.5 This method utilized three
the child’s paternal allele at locus B. to five probes to analyze three to five loci on the same
One repeat unit

GTTCTAGCGGCCGTGGCAGCTAGCTAGCTAGCTGCTGGGCCGTGG
CAAGATCGCCGGCACCGTCGATCGATCGATCGACGACCCGGCACC
FIGURE 10.6 A tandem repeat is a direct repeat of 1
to more than 100 nucleotides in length. The one Tandem repeat (4 units)
shown has a 4-bp repeat unit (AGCT). A gain or loss
of repeat units forms a different allele. Different
GTTCTAGCGGCCGTGGCAGCTAGCTAGCTGCTGGGCCGTGG
alleles are detected as variations in fragment size on CAAGATCGCCGGCACCGTCGATCGATCGACGACCCGGCACC
digestion with a restriction enzyme, such as HaeIII
(GGCC recognition sites). Tandem repeat (3 units)
blot. Results of probing multiple loci at once produced clear results. After visually inspecting the band pat-
patterns that were highly variable between individuals terns, profiles were subjected to computer analysis to
but that required some expertise to optimize and inter- accurately size the restriction fragments and apply the
pret. In 1990, single-locus probe (SLP) systems were results to an established matching criterion. RFLP is an
established in Europe and North America.6,7 Analysis of example of a continuous allele system in which the
one locus at a time yielded simpler patterns, which were sizes of the fragments define alleles. Therefore, precise
much easier to interpret, especially in cases where spec- band sizing was critical to the accuracy of the results. A
imens might contain a mixture of DNA from more than match implied inclusion, which was refined by determi-
one individual (Fig. 10.7). nation of the genotype frequency of each allele in the
The RFLP Southern blot technique required 100 ng general or local population. This process established the
to 1 μg of relatively high-quality DNA, 1 to 20 kbp likelihood of the same genotype occurring by chance.
in size. Furthermore, large, fragile 0.7% gels were The probability of two people having the same set of
required to achieve adequate band resolution, and the RFLP, or profile, becomes lower and lower as more loci
32
P-based probe system could take 5 to 7 days to yield are analyzed.
M 1 P C 2 M E M 1 P C 2 M E
Professor Sir Alec John Jeffreys, a British genet-
icist, first developed techniques for genetic pro-
filing, or DNA fingerprinting, using RFLP to
identify humans. The technique has been used in
forensics and law enforcement to resolve paternity
and immigration disputes. The method can also
be applied to nonhuman species, for example, in
wildlife population genetics. The first application
of this DNA technique was in a regional screen
of human DNA to identify the rapist and killer
of two girls in Leicestershire, England, in 1983
and 1986. Colin Pitchfork was identified and
convicted of murder after samples taken from
him matched semen samples taken from the two
victims.
FIGURE 10.7 Example of RFLP crime evidence using two STR TYPING BY PCR
single-locus probes. M denotes molecular-weight markers, 1
and 2 are suspects, C is the child victim, and P is the parent of
the child victim. E is evidence from the crime scene. For both
The first commercial and validated typing test based on
loci probed, suspect 2 “matches” the evidence found at the polymerase chain reaction (PCR) specifically for forensic
crime scene. Positive identification of suspect 2 requires use was the HLA DQ alpha system, now called DQA1,
further determination of the frequencies of these specific developed in 1986.8 This system could distinguish
alleles in the population and the probability of matching them 28 DQA1 types. With the addition of another commer-
by chance. cial system, the Polymarker (PM) system, the analyst
could type five additional genetic markers. The PM

system is a set of primers complementary to sequences Advanced Concepts
flanking STRs, or microsatellites. STRs are similar
to VNTRs (minisatellites) but have repeat units of 1 Although STRs with 4- and 5-bp repeat units are
to 7 bp. (The upper limit of repeat unit size for STR highly informative and efficiently amplified, they
varies from 7 to 10 bp, depending on different texts and are subject to naturally occurring genetic events.
reports.) Because of the increased power of discrimi- Loss or gain of repeats or parts of repeat units,
nation and ease of use of STR, the HLA DQA foren- as well as mutations within repeat units, are very
sic DNA amplification and typing kit was discontinued rare occurrences. Because at least 8 to more than
in 2002. 20 loci are included in STR applications, these
The tandem repeat shown in Figure 10.6 is an STR minor events do not significantly affect the infor-
with a 4-bp repeat unit, AGCT. Occasionally, STRs mative power. (Allele population frequency has
contain repeat units with altered sequences, or micro- the most limiting effect on inclusion.) In abnormal
variants, repeat units missing one or more bases of the cells with genetic instability, such as cancer cells,
repeat. These differences have arisen through mutation gain or loss of repeats can occur more frequently,
or recombination events. enough to affect the identification of genotypes.10
In contrast to VNTRs, the smaller STRs are effi-
ciently amplified by PCR, easing specimen demands
significantly. Long, intact DNA fragments are not STR alleles are identified by PCR product size. Primers
required to detect the STR products; therefore, degraded are designed to produce amplicons of 100 to 400 bp in
or otherwise less-than-optimal specimens are potentially which the STRs are embedded (Fig. 10.8). The sizes
informative. The amount of specimen required for STR of the PCR products are influenced by the number of
analysis by PCR is reduced from 1 μg to 10 ng, a key embedded repeats. If one of each primer pair is labeled
factor for forensic analysis. Furthermore, PCR proce- with a fluorescent marker, the PCR product can be ana-
dures shorten the analysis time from several weeks to lyzed in fluorescent detection systems. Silver-stained
24 to 48 hours. Careful design of primers and ampli- gels may also be used; however, capillary electrophore-
fications facilitated multiplexing and automation of the sis with fluorescent dyes is the preferable method, espe-
process.9 cially for high-throughput requirements.
Allele 1
TH01
…TCATTCATTCATTCATTCATTCATTCATTCAT…
…AGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTA…
Allele 2
FIGURE 10.8 STR TH01 (repeat unit TCAT) linked to
the human tyrosine hydroxylase gene on chromosome
11p15.5. Primers are designed to amplify short regions …TCATTCATTCATTCATTCATTCATTCATTCATTCAT…
containing the tandem repeats. Allelic ladders consisting of …AGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTAAGTA…
all alleles in the human population (flanking lanes in the
gel shown at bottom right) are used to determine the PCR products: 7/8 7/10
number of repeats in the locus by the size of the amplicon. Allele 1 = 187 bp (7 repeats) –11
The two alleles shown contain seven and eight repeats. If Allele 2 = 191 bp (8 repeats)
these alleles were found in a single individual, that person
would be heterozygous for TH01 with a genotype of 7/8.
Compare the 7/8 genotype pattern with the 7/10 genotype
–5
gel pattern.
is inherited as a single haplotype, paternally related men

Histooricaal Higghlligghtts share all Y loci.13
At least three to seven RFLP probes were orig-
inally required to determine genetic identity. STR Analysis
Available probes included G3, MS1, MS8, MS31, To identify STR alleles, test DNA is mixed with the
and MS43, which were subclones of Jeffreys’s primer pairs, buffer, and polymerase to amplify the test
multilocus probes 33.6 and 33.15 and pYNH24m, loci. Primer pairs may be laboratory designed or pur-
MS205, and MS621. SLPs MS1, MS31, MS43, chased commercially. A control DNA standard is also
G3, and YNH24 were used in the O. J. Simpson amplified, as well as a sensitivity control, if the rela-
trial in 1996. tive allele percentage in a mixture will be calculated.
Following amplification, each sample PCR product is
combined with allelic ladders (sets of fragments repre-
senting all possible alleles of a repeat locus) and internal
A further development of STR analysis was the design of size standards (molecular-weight markers) in formamide
mini-STR. These STRs are amplified with PCR primers for electrophoresis. After electrophoresis, detection and
located closer to the tandem repeat than in the standard analysis software will size and identify the alleles based
STR. Compared with standard STR products, the small on co-size migration with specific alleles in the allelic
amplicons are more efficiently produced from such chal- ladders. In contrast to RFLPs and VNTRs, STRs are dis-
lenging starting material as fixed tissue11 and degraded crete allele systems in which a finite number of alleles
specimens.12 Another specialized system, Y-STR, was is defined by the number of repeat units in the tandem
developed for surname testing and forensic identification repeat (see Fig. 10.8). Several available commercial
of male offenders or victims. This primer set only ampli- systems consist of labeled primers for 1 to more than
fies STR located on the Y chromosome. There is only 16 loci. The allelic ladders in these reagent kits allow
one allele at each locus, and because the Y chromosome accurate identification of the sample alleles (Fig. 10.9).14
FGA PentaE
TPOX D18S51
D8S1179 D2S11
TH01
FIGURE 10.9 Multiple STRs can be re-
solved on a single gel. Here, four and five
different loci are shown on the left and right
gels, respectively. The allelic ladders show
that the ranges of potential amplicon sizes
vWA D3S1358 do not overlap, allowing resolution of multi-
ple loci in the same lane. Two individual
genotypes are shown on the second and
third lanes of the two gels.
STR by gel electrophoresis

Advanced Concepts
–11
Theoretically, the minimal sample requirement
for PCR analysis is a single cell. A single cell
has approximately 6 pg of DNA. This number is
–5
derived from the molecular weight of A/T and
G/C base pairs (617 and 618 g/mol, respectively).
STR by capillary electrophoresis
There are about 3 billion base pairs in one copy
of the human genome; therefore, for one genome 7 8
copy:
3 × 109 bp × 618 g mol bp = 1.85 × 1012 g mol
1.85 × 1012 g mol × 1 mol 6.023 × 1023 molecules 7 10
= 3.07 × 10−12 g = 3 pg
(A diploid cell has two genome copies, or 6 pg of
DNA.) One ng (1,000 pg) of DNA should, there-
fore, contain 333 copies (1,000 pg/3 pg/genome
copy) of each locus.
5 11
Advances in fluorescence technology have increased FIGURE 10.10 STR analysis by capillary gel electrophore-
the ease and sensitivity of STR allele identification sis. Instead of bands on a gel (top), peaks of fluorescence on an
(Fig. 10.10). Although capillary electrophoresis is faster electropherogram reveal the PCR product sizes (bottom).
and more automated than gel electrophoresis, a single Alleles (7, 8, or 10) are determined by comparison with allelic
run through a capillary of single dye-labeled products ladders representing all possible alleles (from 5 to 11 repeats)
for this locus, run through the capillary simultaneously with
can resolve only loci whose allele ranges do not overlap.
the sample amplicons.
The number of loci that can be resolved on a single
run was increased by the use of multicolor dye labels.
Primer sets labeled with dyes that can be distinguished 100 bp 200 bp 300 bp 400 bp
by their emission wavelength generate products that are
resolved according to fluorescent color as well as size D3S1358 vWA FGA Penta E
(Fig. 10.11). Test DNA amplicons, allelic ladders, and D8S1179 D21S11 D18S1179 Penta D
size standards for multiple loci are thus run simultane-
ously through each capillary. Genotyping software pro- D5S818 D13S317 D7S820
vides automated resolution of fluorescent dye colors and

FIGURE 10.11 An illustration of the ranges of allele peak
genotyping by comparison with the size standards and locations for selected STRs. By labeling primers with different
the allelic ladder. fluorescent dye colors, STRs with overlapping size ranges can
be resolved by color. The molecular-weight markers (bottom)
are labeled with a fluorescent dye distinguishable from those
Advanced Concepts used for the primer labeling.
Commercial primer sets are designed with

“stuffer” sequences to modify the size of the PCR the product size for a given allele will not always
products so that the range of alleles for four to five be the same with primers from different commer-
loci can be resolved by electrophoresis. Therefore, cial sources.
As in RFLP testing, an STR “match” is made by com- Analysis of Test Results

paring profiles (alleles at all loci tested) followed by
Analysis of polymorphisms at multiple loci results in
probability calculations. The HLA DQ in conjunction
very high levels of discrimination (Table 10.3). Dis-
with the PM system generated highly discriminatory
covery of the same set of alleles from different sources
allele frequencies. For example, the chance of the same
or shared alleles between allegedly related individuals
set of alleles or profile occurring in two unrelated indi-
is strong evidence of identity, paternity, or relatedness.
viduals at random is 1 in 106 to 7 × 108 Caucasians or
Results from such studies, however, must be expressed in
1 in 3 × 106 to 3 × 108 African Americans.
terms of the background probability of chance matches.
STR Nomenclature
The International Society for Forensic Genetics rec- Histooricaal Higghlligghtts
ommended nomenclature for STR loci in 1997.15 STRs
within genes are designated according to the gene name; The GDB is overseen by the Human Genome
for example, the STR TH01 is located in intron 1 of the Nomenclature Committee, a part of the Human
human tyrosine hydroxylase gene on chromosome 11, Genome Organization (HUGO) located at Univer-
and TPOX is located in intron 10 of the human thyroid sity College, London. HUGO was established in
peroxidase gene on chromosome 2. These STRs do not 1989 as an international association of scientists
have any phenotypic effect with respect to these genes. involved in human genetics. The goal of HUGO
Non-gene-associated STRs are designated by the D#S# is to promote and sustain international collabora-
system. In this system, the “D” stands for DNA; the fol- tion in the field of human genetics. The GDB was
lowing number designates the chromosome where the originally used to organize mapping data during
STR is located (1-22, X or Y). “S” refers to a unique the earliest days of the Human Genome Project.
segment, followed by a number registered in the Inter- With the release of the human genome sequences
national Genome Database (GDB). See Table 10.2 for and the development of PCR, the number of lab-
some examples. oratories doing genetic testing grew significantly.
STRs are present all over the genome. Some of the The GDB is still widely used as a source of infor-
STR loci commonly used for laboratory investigation mation about PCR primers, PCR products, poly-
are shown in Table 10.2. A comprehensive collection of morphisms, and genetic testing. Information from
STR information is available at http://www.cstl.nist.gov/ the GDB is available at http://www.ncbi.nlm.nih
strbase/. .gov/sites/genome.
Gender Identification
The amelogenin locus is a very useful marker often ana-
lyzed along with STR. The amelogenin gene, which is bp: 140 150 160 170 180 190 200 210 220 230 240 250
not an STR, is located on the X and Y chromosomes. The
function of its encoded protein is required for embryonic
development and tooth maturation. A polymorphism is
located in the second intron of the amelogenin gene. The
Y allele of the gene is 6 bp larger in this region than in
the X allele. Amplification and electrophoretic resolu-
tion reveal two bands or peaks for males (XY) and one
band or peak for females (XX; Fig. 10.12). Some com-
mercially available sets will contain primers to amplify FIGURE 10.12 Amplification of amelogenin will produce a
the amelogenin polymorphism in addition to the STR male-specific 218-bp product (Y allele) in addition to the
primer sets. Additional loci are now available for gender 212-bp product found on the X chromosome (X allele). Males
testing, in cases where amelogenin may be compromised are heterozygous for the amelogenin locus (XY, top), and
or poorly amplified.16 females are homozygous for this locus (XX, bottom).
TABLE 10.2 STR Locus Information*71
Chromosome
STR Locus Sequence Repeat Alleles†
CD4 Locus between CD4 and 12p AAAAG‡ 4, 6, 7, 8, 8’, 9, 10, 11, 12, 13, 14, 15
triosephosphate isomerase
CSF1PO c-fms protooncogene for 5q TAGA 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
CSF-1 receptor
D3S1358 3p TCTA§ 8, 9, 10, 11, 12, 13, 14, 15, 15’, 15.2, 16, 16’,
16.2, 17, 17’, 17.1, 18, 18.3, 19, 20
D5S818 5q AGAT 7, 8, 9, 10, 11, 12, 13, 14
D7S829 7q GATA 7, 8, 9, 10, 11, 12, 13, 14, 15
D8S1179 Sequence tagged site 8q TCTA 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
D13S317 13q TATC 7, 8, 9, 10, 11, 12, 13, 14, 15
D16S539 16q GATA 5, 8, 9, 10, 11, 12, 13, 14, 15
D18S51 Sequenced tagged site 18q GAAA 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27
D21S11|| 21q TCTG 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38
F13A01 Coagulation factor IX 6p GAAA 3.2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
F13B Factor XIII b 1q TTTA 6, 7, 8, 9, 10, 11, 12
FESFPS c-fes/fps protooncogene 15q ATTT 7, 8, 9, 10, 11, 12, 13, 14
HPRTB Hypoxanthine Xq TCTA 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17
phosphoribosyl-transferase
LPL Lipoprotein lipase 8p TTTA 7, 8, 9, 10, 11, 12, 13, 14
TH01 Tyrosine hydroxylase 11p TCAT 5, 6, 7, 8, 9, 9.3, 10, 11
TPOX Thyroid peroxidase 2p TGAA 6, 7, 8, 9, 10, 11, 12, 13
vWA Von Willebrand factor 12p TCTA 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21
Penta D 21q AAAGA 2.2, 3.2, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17
Penta E 15q AAAGA 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 20.3, 21, 22, 23, 24
*http://www.cstl.nist.gov/
†
Some alleles have units with one, two, or three missing bases.
‡
In an alternate 8-repeat allele, one repeat sequence is AAAGG.
§
In alternate 15-, 16-, or 17-repeat alleles, one repeat sequence is TCTG.
||
D21S11 has multiple alternate alleles.
TABLE 10.3 Matching Probability of STR 95 100 105 110 115 120 125 130 135 140 145
Genotypes in Different Subpopulations
African White Hispanic

American American American
15 15.2
8 loci 1/274,000,000 1/114,000,000 1/145,000,000
9 loci 1/5.18 × 109 1/1.03 × 109 1/1.84 × 109
10 loci 1/6.76 × 1010 1/9.61 × 1010
12 loci 1/4.61 × 1012 12 13 14 15 16 17 18 19
14 loci 1/6.11 × 1017 1/9.96 × 1017 1/1.31 × 1017 FIGURE 10.13 A microvariant allele (15.2, top) migrates
between the full-length alleles (15 and 16 on the allelic ladder,
16 loci 1/7.64 × 1017 1/9.96 × 1017 1/1.31 × 1017 bottom).
chromosomes from an individual, the locus would be

heterozygous, with 7 repeats on one chromosome and
8 repeats on the other. This locus would thus be des-
Advanced Concepts ignated 7/8 or 7,8. A homozygous locus (where both
homologous chromosomes carry the same allele) is
In 1997 the Federal Bureau of Investiga-
designated by the 7/7 or 7,7. Some reports use a single
tion adopted 13 “core” loci as the Combined
number, such as 6 or 7, to designate a homozygous locus.
DNA Indexing System (CODIS). The loci are
Microvariant alleles containing partial repeat units are
TPOX on chromosome 2, D3S1358 on chromo-
indicated by the number of complete repeats followed by
some 3, FGA on chromosome 4, D5S818 and
a decimal point and then the number of bases in the partial
CSF1PO on chromosome 5, D7S820 on chro-
repeat. For example, the 9.3 allele of the TH01 locus has
mosome 7, D8S1179 on chromosome 8, TH01
10 repeats, 9 full 4-bp repeat units and 1 repeat unit with
on chromosome 11, vWA on chromosome 12,
3 bp. Microvariants are detected as bands or peaks very
D13S317 on chromosome 13, D16S539 on chro-
close to the full-length allele (Fig. 10.13).
mosome 16, D18S51 on chromosome 18, D21S11
The genotype, or profile, of a specimen is the col-
on chromosome 2, and the amelogenin locus on
lection of alleles in all the loci tested. To determine the
the X and Y chromosomes. The National Institute
extent of certainty that one profile matches another, the
of Standards and Technology supplies Standard
occurrence of the detected genotype in the general or a
Reference Material that certifies values for 22
defined population must be assessed.
STR loci, including CODIS and markers used by
A matching genotype is not necessarily an absolute
European forensic laboratories.
determination of the identity of an individual. Genetic
concordance is a term used to express the situation
where all locus genotypes (alleles) from two sources are
the same. Concordance is interpreted as inclusion of a
Genotyping
single individual as the donor of both genotypes. Two
DNA testing results in peak or band patterns that must samples are considered different if at least one locus
be converted to genotype (allele identification). As genotype differs (exclusion). An exception is paternity
described previously, an STR locus genotype is defined testing, in which mutational events may generate a new
by the number of repeats in its alleles. For instance, if the allele in the offspring at one locus, and this difference
locus genotype in Figure 10.8 represented homologous may not rule out paternity.
Technical artifacts such as air bubbles, crystals, and sizes can be established. An acceptable range of sizes
dye blobs, as well as sample contaminants, temperature in this distribution is a bin. A bin can be thought of as
variations, and voltage spikes, can interfere with consis- an uncertainty window surrounding the mean position
tent band migration during electrophoresis. In addition, (size) of multiple runs of each peak or band. All bands
amplification artifacts occur during PCR. Some poly- or peaks, therefore, that fall within this window are con-
merases add an additional non-template adenine residue sidered identical. Collection of all peaks or bands within
to the 3′ end of the PCR product. If this 3′ nucleotide a characteristic distribution of positions and areas is
addition does not include all the amplified products, called binning. Bins for each allele can be established
a mixed set of amplicons will result in extra bands or manually in the laboratory. Alternatively, commercially
peaks located very close together. available software has been designed to automatically
Stutter is another anomaly of PCR amplification, bin and identify alleles.17
in which the polymerase may miss a repeat during the All peaks within a bin are interpreted as representa-
replication process, resulting in two or more different tive of the same allele of a locus. Each band or peak in a
species in the amplified product. These also appear as genotype is binned and identified according to its migra-
extra bands or peaks. Generally, the larger the repeat tion characteristics. The group of bands or peaks makes
unit length, the less stutter is observed. These or other up the characteristic pattern or profile of the specimen.
aberrant band patterns confuse the analysis software and
can result in the miscalling of alleles.
Matching requires clear and unambiguous laboratory Advanced Concepts
results. As alleles are identified by gel resolution, good
intragel precision (comparing bands or peaks on the Binning may be performed in different ways using
same gel or capillary) and intergel precision (compar- replicate peak heights and positions. To calculate
ing bands or peaks of separate gels or capillaries) are the probability that two peaks are representative
important. In general, intergel precision is less stringent of the same allele, the proportion of alleles that
than intragel precision. This is not unexpected because fall within the uncertainty window (bin) must be
the same samples may run with slightly different migra- determined. This proportion is represented exactly
tion speeds on different gels. Because some microvariant in fixed bins and approximated in floating bins.
alleles differ by only a single base pair (see Fig. 10.13), The fixed-bin approach is an approximation of
the resolution must be less than ±0.5 bp. The TH01 9.3 the more conservative floating-bin approach.18 An
allele described earlier is an example. This allele must alternative assessment of allele certainty is the
be distinguished from the 10 allele, which is a single use of locus-specific brackets. In this approach,
base pair larger than the 9.3 allele. artificial “alleles” are designed to run at the high
To establish the identity of peaks from capillary elec- and low limit of the expected allele size. Identi-
trophoresis (or peaks from densitometry tracings of gel cal alleles are expected to fall within this defined
data), the peak is assigned a position relative to some bracket.17
landmark within the gel lane or capillary, such as the
loading well or the start of migration. Upon replicate
resolutions of a band or peak, electrophoretic variations
Matching of Profiles
from capillary to capillary, lane to lane, or gel to gel
may occur. Normalization of migration is achieved by The number of loci tested must be taken into consid-
the relation of the migration of the test peaks to the eration in genotyping analysis. The more loci analyzed,
simultaneous migration of size standards. Size stan- the higher the probability that the locus genotype posi-
dards can be internal (in the same gel lane or capillary) tively identifies an individual (match probability; see
or external (in a separate gel lane). Even with normal- Table 10.3). Degraded, compromised, or mixed samples
ization, however, tiny variations in position, height, will affect the match probability because all loci may
and area of peaks or gel bands may persist. If the same not yield clear, informative results. Criteria for interpre-
fragments are run repeatedly, a distribution of observed tation of results and determination of a true allele are
established by each laboratory. These criteria are based Individual allele frequencies are determined by data
on validation studies and results reported from other collected from testing many individuals in general and
laboratories. Periodic external proficiency testing is per- defined populations. For example, at locus Penta D on
formed to confirm the accuracy of test performance. chromosome 21, the 5 allele has been previously deter-
Results from the analysis of polymorphisms are used mined to occur in 1 of 10 people in a theoretical popu-
to determine the probability of identity or inheritance lation. At locus D7S829 on chromosome 7, the 8 allele
of genetic markers or to match a particular marker or has been previously observed in 1 of 50 people in the
marker pattern. To establish the identity of an individual same population. The overall frequency of the profile
by an allele of a locus, the chance that the same allele containing the loci Penta D 5 allele and D7S829 8 allele
could arise in the population randomly is taken into would be 1/10 × 1/50 = 1/500. That is, a genotype or
account. profile containing D7S829 8 and Penta D 5 alleles would
be expected to occur in 1 out of every 500 randomly
chosen members of that population. As should be appar-
Advanced Concepts ent, the more loci tested, the greater the certainty that the
profile is unique to a single individual in that population;
The certainty of a matching pattern increases with
that is, the overall frequency of the profile is very low.
decreased frequency of alleles in the general pop-
The overall frequencies in Table 10.3 illustrate this point.
ulation. Under defined conditions, the relative
Allele frequencies differ between subpopulations
frequency of two alleles in a population remains
or ethnic groups. Different allele frequencies in sub-
constant. This is Hardy–Weinberg equilibrium,
populations are determined through the study of each
or the Hardy–Weinberg law.19 The population fre-
ethnic group.20 The data in Table 10.3 illustrate differ-
quency of two alleles, p and q, can be expressed
ences in the polymorphic nature of alleles in different
mathematically as
subpopulations.
p2 + 2pq + q 2 = 1.0 When profile identification requires comparing the
This equilibrium assumes a large population with genotype of an unknown specimen with a known ref-
random mating and no immigration, emigration, erence sample—for example, the genotype of evidence
mutation, or natural selection. Under these circum- from a crime and the genotype of an individual from
stances, if enough individuals are assessed, a close a database—the determination that the two genotypes
approximation of the true allele frequency in the match (are from the same person) is expressed in terms
population can be determined. of a likelihood ratio. The likelihood ratio is the compar-
ison of the probability that the two genotypes came from
the same person with the probability that the two geno-
The frequency of a set of alleles or a genotype in a pop- types came from different persons, taking into account
ulation is the product of the frequency of each allele allele frequencies and linkage equilibrium in the pop-
separately (the product rule). The product rule can be ulation. A high likelihood ratio is an indication that the
applied because of linkage equilibrium. Linkage equilib- probability is more likely that the two genotypes came
rium assumes that the observed frequencies of haplotypes from the same person, whereas a likelihood ratio of less
in a population are the same as haplotype frequencies than 1 indicates that this probability is less likely. If a
predicted by multiplying together the frequency of indi- likelihood ratio is [1/(1/1,000)] = 1,000, the tested geno-
vidual genetic markers in each haplotype and that loci types are 1,000 times more likely to have come from the
are not genetically linked (located close to one another) same person than from two randomly chosen members
in the genome. The overall frequency (OF) of a locus of the population, where the profile occurs in 1/1,000
genotype consisting of n loci can be calculated as people. In a random sampling of 100,000 members of
a population, 100 people (100,000/1,000) with the same
OF = F1 × F2 × F3 × . . . Fn genotype might be found.
where F1 . . . n represents the frequency of each individual A simplified illustration can be made from the previ-
allele in the population. ous Penta D and D7S829 example. Suppose the Penta
D 5 (1/10 allele frequency) and D7S829 8 (1/50 allele differently defined groups. It is also important to con-
frequency) profiles were discovered in a specimen from sider whether the population is homogeneous (a random
an independent source. The likelihood that the Penta mixture) with respect to the alleles tested. Familial
D 5/D7S829 8 profile came from the tested individual is searches and forensic applications involving mass disas-
1, having been directly determined. The likelihood that ters or other complex mixtures of DNA involve analysis
the same profile could come from someone else in the of partial or uncertain DNA genotypes. More advanced
population is 1/500. The likelihood ratio is 1/(1/500), or approaches, such as “wild card” designations for missing
500. The specimen material is 500 times more likely to alleles, are required for defining the likelihood in these
have come from the tested individual than from some cases.21
other person in the population.
Allelic Frequencies in Paternity Testing
Histooricaal Higghlligghtts A paternity test is designed to choose between two
hypotheses: The test subject is not the father of the tested
Sir Alec Jeffreys’ DNA profiling was the basis for
child (H0), or the test subject is the father of the tested
the National DNA Database (NDNAD) launched
child (H1). Paternity is first assessed by observation of
in Britain in 1995. Under British law, the DNA
shared alleles between the alleged father and the child
profile of anyone convicted of a serious crime is
(Fig. 10.14). The identity of shared alleles is a process
stored on a database. This database includes 6%
of matching, as described previously for identity testing.
of the UK population (compared with 0.5% of the
population in the U.S. database). Over 5.9 million
DNA profiles are held in the database, accounting Histooricaal Higghlligghtts
for a majority of the known active-offender pop-
ulation. Nearly 60% of crime-scene profiles sub- Peter Gill developed a forensic DNA identifi-
mitted to the NDNAD were matched to a subject cation method for minimal samples called low-
profile between 2008 and 2009. copy-number analysis.22 In contrast to standard
The National DNA Index System (NDIS) DNA analysis that requires approximately 200 pg
is the federal level of the CODIS used in the DNA, low-copy-number analysis is reportedly
United States. There are three levels of CODIS: performed on less than 100 pg DNA (about
the Local DNA Index System (LDIS), State DNA 16 diploid cells). The method involves increas-
Index System (SDIS), and NDIS. At the local ing the number of PCR cycles for amplification
level, CODIS software maintained by the Federal and fastidiously cleaned work areas. Although
Bureau of Investigation (FBI) is used in sizing used in over 21,000 serious criminal cases since
alleles as the assay is performed. This informa- the 1990s, the validity of this technique has been
tion may be compiled locally and/or submitted questioned in appeal cases.23 Assaying limited
to the SDIS. The state data may be sent to the amounts of starting material may result in peak
NDIS. The SDIS and NDIS must adhere to the dropouts (failed amplification of an allele) or
quality assurance standards recommended by the drop-ins (mis-priming of an allele). Further-
FBI. The original entries to these databases were more, the heightened detection limit required
RFLP profiles; further entries have been the STR for low amounts of target DNA raises the risk of
profiles. As of October 2018, the NDIS contained contamination.
over 13 million offender profiles.
Because each allele of a genotype is inherited from one

When comparing genotypes with those in a database parent, a child will share one allele of every locus with
looking for a match, it is important to consider whether the paternal parent. A paternity index, or likelihood
the database is representative of a population or subpop- of paternity, is calculated for each locus in which the
ulation because allele frequencies will be different in alleged father and the child share an allele. The paternity
vWA TH01 AMEL TP0X F13A01 CSF

M
FIGURE 10.14 Electropherogram showing results from

F five STR loci and the amelogenin locus for a child (C),
mother (M), and father (F). Note how the child has inher-
ited one of each allele from the mother (black dots) and
one from the father.
and evaluates the genotype information. The CPI for the

TABLE 10.4 Example Data From a Paternity Test data shown in Table 10.4 are
Showing Inclusion
CPI = 5.719 × 8.932 × 15.41 × 10.22 = 8, 044.931
Father Shared Paternity These data indicate that the child is 8,045 times more
Alleged Child Allele Allele Index likely to have inherited the four observed alleles from the
alleged father than from another man in the population.
D16S539 8, 9 9, 10 9 5.719 If a paternal allele does not match between the alleged
D5S818 10, 12 7, 12 12 8.932
father and the child, H1 for that allele is 0. One might
assume, therefore, that the nonmatching allele paternity
FESFPS 9, 13 13, 14 13 15.41 index of 0 would make the CPI 0. This is not the case.
F13A01 4, 5 5, 7 5 10.22
Nonmatching alleles between the alleged father and the
child found at one locus (exclusion) is traditionally not
regarded as a demonstration of non-paternity because
of the possibility of mutation. Although mutations were
index is an expression of how many times more likely
quite rare in the traditional RFLP systems, analysis of
the child’s allele is inherited from the alleged father than
12 or more STR loci may occasionally reveal one or two
by another man in the general population. An allele that
mutations resulting in nonmatching alleles even if the
occurs frequently in the population has a low paternity
man is the father. To account for mutations, the paternity
index; a rare allele has a high paternity index. Table
index for nonmatching alleles is calculated as
10.4 shows the paternity index for each of four loci. The
FESFPS 13 allele is rarer than the D 16S539 9 allele. In paternity index for a mutant allele = μ
this example, the child is 5.719 times more likely to have where μ is the observed mutation rate (mutations/mei-
inherited the 9 allele of locus D16S539 from the alleged osis) of the locus. The American Association of Blood
father than from another random man in the population. Banks (AABB) has collected data on mutation rates in
Similarly, the child is 15.41 times more likely to have STR loci (Table 10.5). Using these data, in the case of a
inherited the 13 allele of FESFPS from the alleged father nonmatching allele, H1 is not 0 but μ. A high mutation
than by random occurrence. When each tested locus rate (close to 1) would not lower the CPI, whereas a very
is on a different chromosome (not linked), the inheri- low mutation rate (closer to 0) would do so.
tance or occurrence of each allele can be considered an In a paternity report, the combined paternity index is
independent event. The paternity index for each locus, accompanied by the probability of paternity, a number
therefore, can be multiplied together to calculate the calculated from the combined paternity index (genetic
combined paternity index (CPI), which summarizes evidence) and prior odds (nongenetic evidence). For the
TABLE 10.5 Observed Mutation Rates TABLE 10.6 Odds of Paternity Using Different
in Paternity Tests Using STR Loci Prior Odds Assumptions
STR Locus Mutation Rate (%) Prior Odds
D1S1338 0.09 CPI 10% 25% 50% 75% 90%

D3S1358 0.13
5 0.36 0.63 0.83 0.94 0.98
D5S818 0.12
9 0.50 0.75 0.90 0.96 0.98
D7S820 0.10
19 0.68 0.86 0.95 0.98 0.994
D8S1179 0.13
99 0.92 0.97 0.99 0.997 0.999
D13S317 0.15
999 0.99 0.997 0.999 0.9997 0.9999
D16S539 0.11
D18S51 0.25 In the example illustrated previously, the CPI is

8,044.931. The probability of paternity is
D19S433 0.11
8, 044.931 × 0.50
= 0.999987
D21S11 0.21 (8, 044.931 × 0.50) + 0.50
CSF1PO 0.16 The genetic evidence (CPI) has changed the probability
of paternity (prior odds) of 50% to 99.9987%.
FGA 0.30
There is some disagreement about the assumption of
TH01 0.01 50% prior odds. Using different prior odds assumptions
changes the final probability of paternity (Table 10.6).
TPOX 0.01
As can be observed from the table, however, at a CPI
VWA 0.16 over 100, the differences have less effect.
F13A01 0.05 Sibling Tests
FESFPS 0.05 Polymorphisms are also used to generate a probability of
F13B 0.03 siblings or other blood relationships (familial searches).24
A sibling test is a more complicated statistical analysis
LPL 0.05 than a paternity test. Mutations and allele frequencies
Penta D 0.13 further complicate analysis.25 More confident conclusions
can be made with multiple siblings. Methods involving
Penta E 0.16 parental genotype reconstruction have been proposed.26
A full sibling test is a determination of the likelihood that
two people tested share a common mother and father. A
prior odds, the laboratory as a neutral party assumes a half-sibling test is a determination of the likelihood that
50/50 chance that the test subject is the father. There- two people tested share one common parent (mother or
fore, the probability of paternity is father). The likelihood ratio generated by a sibling test
CPI × prior odds is sometimes called a kinship index, sibling index, or
combined sibling index.
(CPI × prior odds) + (1 − prior odds)
Another type of relationship analysis is avuncu-
CPI × 0.50 lar testing, which measures the probabilities that two
=
(CPI × 0.50) + (1 − 0.50) alleged relatives are related as either an aunt or an uncle
of a niece or nephew. The probability of relatedness is parent, Y-STRs are represented only once per genome
based on the number of shared alleles between the tested and only in males (Fig. 10.15). A set of Y-STR alleles
individuals. As with paternity and identity testing, allele comprises a haplotype, or series of linked alleles always
frequency in the population will affect the significance inherited together. This is because the Y chromosome
of the final results. The probabilities can be increased cannot extensively exchange information (recombine)
greatly if other known relatives, such as a parent of the with the X chromosome or another Y chromosome.
niece or nephew, are available for testing. Determination Thus, marker alleles on the Y chromosome are inher-
of first- and second-degree relationships is important ited from generation to generation in a single block.
for genetic studies because linkage mapping of disease This means that the frequency of entire Y-STR profiles
genes in populations can be affected by undetected (haplotypes) in a given population can be determined
familial relationships.27 by empirical studies. For example, if a combination of
alleles (haplotype) was observed only two times in a test
of 200 unrelated males, that haplotype is expected to
Y-STR
occur with a frequency of approximately 1 in 100 males
Unlike conventional STRs (autosomal STRs), where tested in the future. The discrimination power of
each locus is defined by two alleles, one from each Y-haplotype testing will depend on the number of
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
Y-PLEX LADDER
12–14 13–16 28–33

Y-PLEX LADDER
22–25 9–12 8, 10–19

Molecular-weight standards
Y alleles
15 15 29
Y alleles
21 10 17
Molecular-weight standards
FIGURE 10.15 Electropherogram showing allelic ladders for six STR loci in the Y-Plex 6 system (top panel) and a single hap-
lotype (bottom panel). Molecular-weight standards are shown at the bottom of each.
subjects tested and will be less definitive than that of Y-specific primers, Y-STR can be specifically amplified
autosomal STR. by PCR from the male–female mixture, resulting in an
Despite being a less powerful system for identifica- analyzable marker that has no female background. This
tion, STR polymorphisms on the Y chromosome have affords a more accurate identification of the male donor.
unique characteristics that have been exploited for foren- The Y chromosome has a low mutation rate. The
sic, lineage, and population studies as well as kinship overall mutation rate for Y chromosome loci is esti-
testing.28 Except for rare mutation events, every male mated at 7.4 × 10−10 mutations per position per year.29
member of a family (brothers, uncles, cousins, and Assuming that Y-chromosome mutations generally
grandfathers) will have the same Y-chromosome haplo- occur once every 500 generations/locus, for 25 loci,
type. Thus, Y-chromosome inheritance can be applied to 1 locus should have a mutation every 20 generations
lineage, population, and human migration studies. The (500 generations/25 markers = 20 generations). Lineage
Y-STR/paternal lineage test can determine whether testing over several generations is made possible by this
two or more males have a common paternal ancestor. low mutation rate. It is also useful for missing persons’
In addition to family history studies, the results of a cases in which reference samples can be obtained from
paternal lineage test serve as supportive evidence for paternally related males.
adoptees and their biological relatives or for individuals A list of informative Y-STRs is shown in Table 10.7.
filing inheritance and benefit claims. Because Y chro- Several Y-STRs are located in regions that are dupli-
mosomes are inherited intact, spontaneous mutations cated on the Y chromosome. DSY389I and DSY389II
in the DNA sequence of the Y chromosome are used to are examples of duplicated loci.
follow human migration patterns and historical lineages. Like autosomal STRs, Y-STRs have microvariant
Y-chromosome genotyping has been used to locate the alleles containing incomplete repeats and alleles con-
geographical origin of populations. taining repeat sequence differences. Reagent systems
Because all male relatives in a family will share the consisting of multiplexed primers for identification of
same allele combination or profile, the statistical signif- 6-17 Y-STR loci are available commercially.
icance of a Y-STR DNA match cannot be assessed by
multiplying likelihood ratios as was described previ-
Matching With Y-STRs
ously for autosomal STR. Instead of the allele frequency
used in autosomal STR match calculations, haplotype Matching probabilities from Y-STR data are deter-
frequencies are used. Estimation of haplotype frequen- mined differently than for the autosomal STR. Haplo-
cies, however, is limited by the number of known Y hap- type diversity (HD) is calculated from the frequency of
lotypes. This smaller data set accounts for the reduced occurrence of a given haplotype in a tested population.
inclusion probabilities and a discrimination rate that is The probability of two random males sharing the same
significantly lower than that for autosomal STR poly- haplotype is estimated at 1-HD; that is, if the haplotype
morphisms. Traditional STR loci are, therefore, preferred diversity is high, the probability of two random males
for identity or relationship analyses, and the Y-STRs are in the population having the same haplotype is low.
used to aid in special situations—for instance, in con- Another measure of profile uniqueness, the discrimina-
firming sibship between males who share commonly tory capacity (DC), is determined by the number of dif-
occurring alleles, that is, have a low likelihood ratio ferent haplotypes seen in the tested population and the
based on traditional STRs. total number of samples in the population. DC expresses
Y-STRs have been utilized in forensic tests where the percentage of males in a population who can be iden-
the evidence consists of a mixture of male and female tified by a given haplotype. Just as the number of loci
DNA, such as semen, saliva, other body secretions, or included in an autosomal STR genotype increases the
fingernail scrapings. For instance, in specimens from power of discrimination, DC is increased by increasing
the evidence of sexual assault, the female DNA may the number of loci defining a haplotype. For instance,
be in vast excess (more than 100-fold) compared to the six loci tested can distinguish 82% of African Ameri-
male DNA in the sample. Autosomal STRs are not con- can males. Using 22 loci raises the DC to almost 99%
sistently informative under these circumstances. Using (Table 10.8).
TABLE 10.7 Y-STR Locus Information*72-74
Y-STR Repeat Sequence† Alleles
DYS19 [TAGA]3TAGG[TAGA]n 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
DYS385 [GAAA]n 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16.3, 17, 17.2 17.3, 18, 19, 20, 21, 22, 23, 24, 28
DYS388 [CAA]n 10, 11, 12, 13, 14, 15, 16, 17, 18
DYS389 I‡ [TCTG]q [TCTA]r 9, 10, 11, 12, 13, 14, 15, 16, 17
DYS389 II‡ [TCTG]n[TCTA]p[TCTG]q[TCTA]r 26, 27, 28, 28’, 29, 29’, 29’’, 29’’’, 30, 30’ 30’’, 30’’’, 31, 31’, 31’’, 32, 32’, 33, 34
DYS390 [TCTG]n[TCTA]m[TCTG]p[TCTA] 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28
DYS391 [TCTA]n 6, 7, 8, 9, 10, 11, 12, 13, 14
DYS392 [TAT]n 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17
DYS393 [AGAT]n 9, 10, 11, 12, 13, 14
DYS426 [CAA]n 6.2, 9, 10, 11, 12, 13, 14
DYS434 [CTAT]n 8, 9, 10, 11
DYS437 [TCTA]n[TCTG]2[TCTA]4 13, 14, 15, 16, 17
DYS438
DYS439 [TTTTC]n 6, 7, 8, 9, 10, 11, 12, 13, 14
DYS439 [GATA]n 9, 10, 11, 12, 13, 14
(Y-GATA-A4) [GATA]n 9, 10, 11, 12, 13, 14
DYS441 [CCTT]n 8, 10.1, 11, 11.1, 12, 13, 13.1, 14, 14.3, 15, 16, 17, 18, 19, 20
DYS442 [TATC]n 8, 9, 10, 11, 12, 12.1, 13, 14, 15
DYS444 [TAGA]n 9, 10, 11, 12, 13, 14, 15, 16
DYS445 [TTTA]n 6, 7, 8, 9, 10, 10.1, 11, 12, 13, 14
DYS446 [AGAGA]n 8, 9, 10, 11, 12, 13, 14, 15, 15.1, 16, 17, 18, 19, 19.1, 20, 21, 22, 23
DYS447 [TTATA]n 15, 16, 17, 18, 19, 19.1, 20, 21, 22, 22.2, 22.4, 23, 24, 25, 26, 26.2, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36
DYS448 [AGAGAT]n 17, 19, 19.2, 20, 20.2, 20.4, 21, 21.2, 21.4, 22, 22.2, 23, 23.4, 24, 24.5, 25, 26, 27
DYS449 [GAAA]n 23, 23.4, 24, 24.5, 25, 26, 27, 27.2, 28, 28.2, 29, 29.2, 30, 30.2, 31, 32, 32.2, 33,
33.2, 34, 35, 36, 37, 37.3, 38
DYS452 [TATAC]n 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35
DYS454 [TTAT]n 6, 7, 8, 9, 10, 11, 12, 13
DYS455 [TTAT]n 7, 8, 9, 10, 11, 12, 13

TABLE 1.7 Y-STR Locus Information (Continued)
Y-STR Repeat Sequence† Alleles
DYS456 [AGAT]n 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
DYS458 [CTTT]n 12, 12.2, 13, 14, 15, 15.2, 16, 16.1, 16.2, 17, 17.2, 18, 18.2, 19, 19.2, 20, 20.2, 21
DYS460 [ATAG]n 7, 8, 9, 10, 10.1, 11, 12, 13

(Y-GATA-A7.1)
*http://www.cstl.nist.gov
†
Some alleles contain repeats with one, two, or three bases missing.
‡
DYS389 I and II is a duplicated locus.
Because there is no recombination between loci on the

TABLE 10.8 Discriminatory Capacity of Y-STR
Y chromosome, the product rule cannot be applied. The
Genotypes in Different Subpopulations75
results of a Y typing might be reported accompanied by
the number of observations or frequency of the analyzed
African White Hispanic
American American American haplotype in a database of adequate size. Suppose a
(%) (%) (%) haplotype containing the 17 allele of DYS390 occurs in
only 23% of men in a database of 12,400. However, if
6 loci* 82.3 68.9 78.3 that same haplotype contains the 21 allele of DYS446,
9 loci† 84.6 74.8 85.1
only 6% of the men will have haplotypes containing
both the DYS390 17 and DYS446 21 alleles. If the
11 loci‡ 91.3 83.8 90.3 11 allele of DYS455 and the 15 allele of DYS458 are
17 loci 99.1 98.8 98.3
also present, only 1 out of 12,400 men in the popu-
lation has a haplotype containing all four alleles. The
20 loci§ 98.5 97.2 98.6 uniqueness of this haplotype is strong evidence that a
|| match is not the result of a random coincidence, which
22 loci 98.9 99.6 99.3
gives extra support to the hypothesis that an independent
*Y-Plex 6 (DYS19, DYS390, DYS391, DYS393, DYS389II, and DYS385). source with this haplotype comes from an individual or
†
European minimal haplotype. a paternal relative. Even with a 1/12,400 or 99.9% DC,
‡
Minimal haplotype + SWGDAM. however, the matching probability is orders of magni-
§
Y-STR 20 plex (minimal haplotype plus DYS388, DYS426, DYS437, DYS439,
DYS460, H4, DYS438 DYS447, and DYS448). tude lower than that for autosomal STR.
||
Y-STR 22 plex. Y-chromosome haplotypes can be used to exclude
paternity. Taking into account the mutation rate of each
Advanced Concepts allele, any alleles that differ between the male child and
the alleged father are strong evidence for non-paternity.
The European Y chromosome typing commu- Conversely, if a Y haplotype is shared between a child
nity has established a set of Y-STR loci termed and alleged father, a paternity index is calculated in a
the minimal haplotype (see http://www.yhrd manner similar to that of the autosomal STR analysis.
.org). The minimal haplotype consists of Y-STR For example, suppose 6 Y-STR alleles are tested and
markers DYS19, DYS389I, DYS389II, DYS390I, match between the alleged father and child. If the haplo-
DYS391, DYS392, DYS393, and DYS385.30 An type has not been observed before in the population, the
“extended haplotype” includes all of the loci occurrence of that haplotype in the population database
from the minimal haplotype plus the highly poly- is 0/1,200, and the haplotype frequency will be 1/1,200,
morphic dinucleotide repeat YCAII. or 0.0008333. The paternity index (PI) is the probability
that an alleged father with that haplotype could produce
one sperm carrying the haplotype, divided by the prob- Family history and analysis of generations of a single
ability that a random man could produce one sperm car- family for the presence of a particular STR allele in
rying the haplotype. The PI is then 1/0.0008333 = 1,200. affected individuals is one way to show association.
With a prior probability of 0.5, the probability of pater- Family members are tested for several STRs, and the
nity is alleles of affected and unaffected members of the family
are compared. Assuming normal Mendelian inheritance,
(1, 200 × 0.5) [(1, 200 × 0.5) + 0.5] , or 99.9%.
if a particular allele of a particular locus is always
This result, however, does not exclude patrilineal rela- present in affected family members, that locus must be
tives of the alleged father. closely linked to the gene responsible for the pheno-
Y-STRs as marker loci for Y-chromosome, or type in those individuals (linkage disequilibrium; Fig.
surname, tests are used to determine ancestry. For 10.16). If the linkage is close enough to the gene (no
example, a group of males of a strictly male descent line recombination between the STR and the disease gene),
(having the same last name or surname) is expected to the STR may serve as a convenient marker for disease
be related to a common male ancestor. Therefore, they testing. Instead of testing for mutations in the disease
should all share the same Y-chromosome alleles (except gene, the marker allele is determined. It is easier, for
for mutations, which should be minimal, given 1 muta- example, to look for a linked STR allele than to screen
tion per 20 generations, as explained previously). The a large gene for point mutations. The presence of the
Y-chromosome haplotype does not provide information “indicator” STR allele serves as a genetic marker for the
about the degree of relatedness, just inclusion or exclu- disease (Fig. 10.17).
sion from a family. An analysis to find a most recent Another approach to linkage analysis is to look for
common ancestor (MRCA) is possible, however, using gene associations in large numbers of unrelated individ-
a combination of researched family histories, Y-STR test uals in population studies. Just as with family history
results, and statistical formulas for mutation frequencies. studies, close linkage to specific STR alleles supports
the genetic proximity of the disease gene with the STR.
In this case, however, large numbers of unrelated people
LINKAGE ANALYSIS are tested for linkage rather than a limited number of
related individuals in a family. The results are expressed
Because the locations of many STRs in the genome are in probability terms that an individual with the linked
known, these structures can be used to map genes, espe- STR allele is likely to have the disease gene. With the
cially those genes associated with disease. Three basic accumulation of genomic data produced by massive par-
approaches are used to map genes: family histories, pop- allel sequencing methods, however, this type of popula-
ulation studies, and sibling analyses. tion study is currently done with higher resolution using
…CACACACA…
FIGURE 10.16 Linkage analysis with
Allele A AB BC
STRs. Three alleles, A, B, and C, of an
STR locus are shown (left). At right is a
family pedigree showing assortment of
the alleles along with gel analysis of PCR
…CACACACACACA…
AC AB BC BB amplification products. Allele C is present
Allele B
in all affected family members. This sup-
ports the linkage of this STR with the
gene responsible for the disease affecting
the family. Analysis for the presence of
Allele C …CACACA… allele C of this STR may also provide a
simple indicator to predict inheritance of
the affected gene.
AB BC
CON BC AC BB AB BC AB BB AC BB AB
FIGURE 10.17 Inheritance of alleles in an

affected family. Using the banding pattern shown, A
the B allele of this STR is always present in B
affected individuals. This locus must be closely
C
linked to the mutated gene.
SNPs, rather than STR (see the following discussion). Autologous bone marrow transplant
Either type of marker is informative if a linkage is found.
Sibling studies are another type of linkage analysis.
Monozygotic (identical) and dizygotic (fraternal) twins
serve as controls for genetic and environmental studies.
Monozygotic twins will always have the same genetic
alleles, including disease genes. There should be 100%
recurrence risk (likelihood) that if one twin has a genetic
disease, the other twin has it, and both should have the Bone marrow cells
same linked STR alleles. Fraternal twins have the same
likelihood of sharing a gene allele as any sibling pair.
Investigation of adoptive families may also distinguish
genetic from environmental or somatic effects. Allogeneic bone marrow transplant
BONE MARROW ENGRAFTMENT TESTING

USING DNA POLYMORPHISMS
Bone marrow transplantation is a method used to treat
malignant and nonmalignant blood disorders as well as
some solid tumors. One transplant approach is autolo-
gous (from self), in which cells from the patient’s own
bone marrow are removed and stored. The patient then
receives high doses of chemotherapy and/or radiother-
FIGURE 10.18 In autologous bone marrow transplant (top),
apy. The portion of marrow previously removed from bone marrow cells are taken from the patient, purged, and
the patient may also be purged of cancer cells before returned to the patient after conditioning treatment. In alloge-
being returned to the patient. Alternatively, allogeneic neic transplant (bottom), bone marrow cells are taken from
transplants (between two individuals) are used. The another genetically compatible individual (donor, right) and
donor supplies healthy cells to the recipient patient given to the patient (left).
(Fig. 10.18). Donor cells are supplied as bone marrow,
peripheral blood stem cells (also called hematopoietic
stem cells), or umbilical cord stem cells. To assure suc-
cessful establishment of the transplanted donor cells,
the immune compatibility of the donor and recipient is
tested prior to the transplant by HLA typing.
database of people who have voluntarily submitted their

Histooricaal Higghlligghtts HLA types and are willing to serve as potential donors.
The first successful bone marrow transplants Stem cells may also be acquired from donated umbil-
were syngeneic transplants; that is, the donor and ical cord blood. After conditioning and infusion with the
recipient pairs were identical twins. In the late donor cells, the patient enters the engraftment phase, in
1950s, compatibility between donor and recipient which the donor cells reconstitute the recipient’s bone
HLAs was found to be necessary for the success marrow. Once a successful engraftment of donor cells
of allogeneic transplants. With the development is established, the recipient is a genetic chimera; that
of genetically defined animals, cross-species or is, the recipient has tissue and blood cells of separate
xenogeneic transplants were considered, and in genetic origins.
1992, a xenotransplant from baboon to human The engraftment of donor cells in the recipient must
was attempted. The patient died 26 days later be monitored, especially in the first 90 days after the
from infection. Currently, donor registries and transplant. This requires distinguishing between donor
advances in the use of hematopoietic stem cells cells and recipient cells. Historically, red blood cell
have broadened the application of transplants for phenotyping, immunoglobulin allotyping, HLA typing,
a variety of diseases. karyotyping, and fluorescence in situ hybridization have
been used for this purpose. Each of these methods has
drawbacks. Some require months before engraftment
In allogeneic transplant strategies, high doses of therapy can be detected. Others are labor-intensive or restricted
completely remove the recipient bone marrow, particu- to sex-mismatched donor–recipient pairs.
larly the stem cells that give rise to all the other cells in DNA typing has become the method of choice for
the marrow (conditioning). The allogeneic stem cells are engraftment monitoring.31,32 Because all individuals,
then expected to reestablish a new bone marrow in the except identical twins, have unique DNA polymor-
recipient (engraftment). The toxicity of this procedure phisms, donor cells are monitored by following donor
was reduced by using sub-myeloablative transplant pro- polymorphisms in the recipient blood and bone marrow.
cedures or mini-transplants. In this approach, pretrans- Although RFLP can effectively distinguish donor and
plant therapy will not completely remove the recipient recipient cells, the detection of RFLP requires the use
bone marrow. The donor bone marrow is expected to of the Southern blot method, which has limited sensi-
eradicate the remaining recipient cells through recog- tivity for this application. In comparison, small VNTRs
nition of residual recipient cells as foreign to the new and STRs are easily detected by PCR amplification of
bone marrow. This process also imparts a graft-versus- VNTRs and STRs (see Fig. 10.9), which is preferable
leukemia (GVL) or graft-versus-tumor (GVT) effect, because of the increased rapidity and the 0.5% to 1% sen-
which is a process closely related to graft-versus- sitivity achievable with PCR. Sensitivity can be raised to
host disease (GVHD). The T-cell fraction of the donor 0.01% using Y-STR, but this approach is limited to those
marrow is particularly important for engraftment and transplants from sex-mismatched donor–recipient pairs,
for GVT effect. This was realized when efforts to avoid preferably from a female donor to a male recipient.
GVHD by removing the T-cell fraction before infusion
of donor cells resulted in increased incidence of graft
failure and relapse. Advanced Concepts
The first phase of allogeneic transplantation is donor
matching, in which potential donors are tested for immu- Chimerism is different from mosaicism. A chimera
nological compatibility. This is performed by examining is an individual carrying two populations of cells
the HLA locus to determine which donor would be most that arose from different zygotes. In a mosaic,
tolerated by the recipient immune system. Donors may be cells arising from the same zygote have undergone
known or related to the patient or anonymous unrelated a genetic event, resulting in two clones of pheno-
contributors (matched unrelated donor [MUD]). The typically different cells in the same individual.
National Marrow Donor Program (NMDP) maintains a
Locus: 1 2 3 4 5 M D R M D R GF MC FC
M D R D R D R D R D R
FIGURE 10.20 Band patterns after PAGE analysis of VNTR.

FIGURE 10.19 Band patterns of five different loci compar- First, before the transplant, several VNTR must be screened to
ing donor (D) and recipient (R) genotypes. The second and find informative loci that differ in pattern between the donor
fifth loci are informative. The first and fourth loci are non- and recipient. One such marker is shown at left (M = molecu-
informative. The third locus is donor-informative. lar-weight marker, D = donor, R = recipient). After the trans-
plant, the band patterns can be used to distinguish between
graft failure (GF), mixed chimerism (MC), or full chimerism
(FC).
In the laboratory, there are two parts to engraftment/
chimerism DNA testing. Before the transplant, several
polymorphic loci in the donor and recipient cells are
screened to find at least one informative locus, that is, Currently, the preferred method is PCR amplification
one locus in which donor alleles differ from the recip- of STRs, resolution by capillary electrophoresis, and
ient alleles. Noninformative loci are those in which fluorescent detection. This procedure provides ease of
the donor and the recipient have the same alleles. In use, accurate quantification of the percentage of donor/
donor-informative loci, the donor and recipient share one recipient cells, and high sensitivity with minimal sample
allele for which the donor is heterozygous and the donor requirements. An alternate, even more sensitive method
has a unique allele. Conversely, in recipient-informative using qPCR of SNP has been proposed.34 This method
loci, the unique allele is in the recipient (Fig. 10.19). also has the advantage of higher throughput and lower
The second part of the testing process is the engraftment sample requirements. It can be performed on a 96-well
analysis, which is performed at specified intervals after plate format as a sequence-specific qPCR with no gel
the transplant. In the engraftment analysis, the recipi- resolution required. Limited informative SNP and the
ent blood and bone marrow are tested to determine the requirement to include pretransplant donor and recipi-
presence of donor cells using the informative and/or ent DNA at each monitoring time point have slowed the
recipient-informative loci. adoption of this method in many laboratories.
Pretransplant analysis and engraftment were mea-
sured in early molecular studies by amplification of
PSTR Testing
small VNTRs and resolution of amplified fragments on
polyacrylamide gels with silver-stain detection.33 Before Donor and recipient DNA for allele screening prior to
the transplant, the screen for informative loci was based transplant can be isolated from blood or buccal cells.
on band patterns of the PCR products, as illustrated in The lower limit of 1 ng of DNA is reportedly sufficient
Figure 10.19. After the transplant, analysis of the gel for the screening of multiple loci; however, 10 ng is a
band pattern from the blood or bone marrow of the more practical lower limit. Multiple loci can be screened
recipient revealed one of three different states: full chi- simultaneously using multiplex PCR. Although not val-
merism, in which only the donor alleles were detected idated for engraftment testing, several systems designed
in the recipient; mixed chimerism, in which a mixture for human identification are used for this purpose. Primer
of donor and recipient alleles was present; or graft sets that specifically amplify Y-STR may also be useful
failure, in which only recipient alleles were detectable for sex-mismatched donor–recipient pairs. Figure 10.21
(Fig. 10.20). shows the five tetramethylrhodamine (TMR)-labeled
loci originally developed for identity testing. Although

multiplex primer systems are optimized for consistent Advanced Concepts
results, all loci may not amplify with equal efficiency in
a multiplex reaction. For example, the amelogenin locus A more defined condition can be uncovered by
in Figure 10.21 did not amplify as well as the other four cell type separation. Some cell fractions such as
loci in the multiplex. This is apparent from the lower granulocytes engraft before others. For example,
peak heights in the amelogenin products compared with isolated granulocytes may show full chimerism,
the products of the other primers. Less efficient amplifi- whereas the T-cell fraction still shows mixed chi-
cation lowers the sensitivity of subsequent post-engraft- merism. This is a case of split chimerism.
ment testing.
Although the capillary electrophoresis used for this
method is the same as that used for sequence analysis,
measuring peak sizes and peak areas is distinguished
vWA TH01 AMEL TPOX CSFIPO
from sequence analysis as fragment analysis and some-
times requires adjustment of the instrument or capillary
polymer. Automatic detection will generate an elec-
tropherogram, as shown in Figure 10.22. Informative
and noninformative loci will appear as nonmatching or
matching donor and recipient peaks, respectively, and
many combinations of donor–recipient peaks are pos-
sible. Optimal loci for analysis should be clean peaks
without stutter, especially stutter peaks that co-migrate
120 140 160 180 200 220 240 260 280 300 320
LPL D5S818 D13S317 D7S820 D16S539
LPL D5S818 D13S317 D7S820 D16S539
vWA TH01 TP0X F13A01 CSF1P0
vWA TH01 TP0X F13A01 CSF1P0
FIGURE 10.22 Screening of 16 loci for informative STR

FIGURE 10.21 Multiplex PCR of DNA mixtures from two alleles. Recipient peak patterns (first and third traces) are com-
unrelated individuals (top and bottom trace) showing peak pat- pared with donor patterns (second and fourth). LPL, D5S818,
terns for vWA, TH01, Amelogenin, TPOX, and CSF1PO loci. D13S317.VWA, THO1, and TPOX are informative. CSF1PO
The center traces are stepwise mixtures of the two genotypes. is donor-informative.
with informative peaks, nonspecific amplified peaks

230 240 250 260 270 280 290 300 310 320
(mis-primes), or other technical artifacts.35 Recipient D16S539
Ideally, the chosen locus should have at least one
recipient informative allele. This is to assure direct detec-
tion of minimal amounts of residual recipient cells. If
the recipient is male and the donor is female, the amelo-
Donor
genin locus supplies a recipient-informative locus. Good
separation (ideally, but not necessarily, by two repeat
units) of the recipient and donor alleles is desirable for
ease of discrimination in the post-transplant testing. The
choices of informative alleles are more limited in related Recipient whole blood
donor–recipient pairs, as they are likely to share alleles.
Unrelated donor–recipient pairs, on the other hand, will
yield more options. 16413 4616
Recipient T-cell fraction
After the transplant, the recipient is tested on a sched-
ule determined by the clinician or according to consen-
sus recommendations.36 The frequency of testing should
be determined by clinical standard operating procedures. 15608 516
Testing will depend on the disease and individual patient
assessment. With nonmyeloablative or reduced-intensity FIGURE 10.23 Post-engraftment analysis of an informative
pretransplant protocols, an example schedule would be locus D16S539. The area (fluorescence units) under the peaks
is calculated automatically. The recipient and donor patterns
testing at 1, 3, 6, and 12 months. Because early patterns
are shown in the first and second trace, respectively. Results
of engraftment may predict GVHD or graft failure after
from the whole blood and T-cell fraction are shown in the third
non-myeloablative treatments, even more frequent blood and fourth traces. For D16S539, the formula R(unshared)/(R(unshared)
testing may be necessary, such as at 1, 2, and 3 months + D(unshared) yields 4,616/(4,616 + 16,413) × 100 = 22% recipi-
after transplant. Bone marrow specimens can most con- ent cells in the unfractionated blood (arrow) and 516/
veniently be taken at the time of bone marrow biopsy (516 + 15,608) × 100 = 3.2% recipient cells in the T-cell
following the transplant, with blood specimens taken in fraction.
intervening periods. Usually, 3 to 5 mL of bone marrow
or 5 mL of blood is more than sufficient for analysis; products as peaks of fluorescence units (y-axis) versus
however, specimens collected soon after the transplant migration speed (x-axis). The amount of fluorescence in
may be hypocellular, so larger volumes (5 to 7 mL bone each product or peak, represented as the height or area
marrow, 10 to 20 mL blood) may be required. under the peak, is used to calculate the percentage of
recipient and donor cells (Fig. 10.23).
Post-Transplant Engraftment Testing
Quantification of the percentage of recipient and donor Advanced Concepts
cells post-transplant is performed using the informative
locus or loci selected during the pretransplant informa- Occasionally, specimens may be received in
tive analysis. The raw data for these calculations are the the laboratory after engraftment without pre-
peak heights or areas under the peaks generated by the engraftment information. In this case, the blood or
PCR products after amplification. Peaks are generated bone marrow of the recipient is not acceptable for
by the emission from the fluorescent dyes attached to determination of recipient-specific alleles because
the primers and thus to the ends of the PCR products the alleles present may represent donor, recipient,
collected as each product migrates past the detector. The or both donor and recipient. The specimen can be
fluorescent signal is converted into fluorescence units by processed using the amelogenin locus or Y-STR
the computer software. The software displays the PCR
markers if the donor and recipient are of differ- monitor graft-versus-tumor potential. T cells may
ent genders, preferably a female donor and a male comprise 10% of peripheral blood leukocytes and
recipient. Another option is to use an alternate 3% of bone marrow cells following allogeneic
source of recipient DNA, such as buccal cells, skin transplantation. Analysis of unfractionated blood
biopsy sample, or stored specimens or DNA from and especially bone marrow where all other lin-
previous testing. Because of the nature of lym- eages are 100% could miss split chimerism in the
phocyte migration, however, skin and buccal cells T-cell fraction. T-cell-lineage-specific chimerism
may also have donor alleles due to the presence will therefore increase the sensitivity of the engraft-
of donor lymphocytes in these tissues. The best ment analysis, particularly after nonmyeloablative
approach is to ensure that informative analysis of and immunoablative pretransplant treatments.
the donor and recipient are part of the pretrans- Testing of the engraftment of lineage-specific
plant agenda. call populations increases the informativity and
sensitivity of the engraftment testing. Selected
cell populations (e.g., T or B lymphocytes, or
There are several formulae for percent calculations,
myeloid cells) are conveniently separated from
depending on the configuration of the donor and recipi-
whole blood using magnetized polymer particles
ent peaks. For homozygous or heterozygous donor and
(beads) attached to cell type–specific antibodies,
recipient peaks with no shared alleles, the percentage
such as pan-T antibodies (anti-CD3) to isolate
of recipient cells is equal to R/(R + D), where R is the
T cells. To isolate cells using beads, whole blood
height or area under the recipient-specific peak(s), and
or bone marrow or white blood cells isolated by
D is the height or area under the donor-specific peak(s).
density-gradient centrifugation are mixed with the
Shared alleles, where one allele is the same for donor
beads in saline or phosphate-buffered saline and
and recipient (Fig. 10.22), can be dropped from the cal-
incubated to allow the antibodies on the beads to
culation, and the percentage of recipient cells is calcu-
bind to the antigens on the cell surface. With the
lated as
beads, cells with bound beads are immobilized by
R ( unshared ) a magnet that is applied to the outside of the tube,
( R ( unshared ) + D( unshared ) ) and the supernatant containing cells without bound
beads is decanted. After another saline wash, the
Chimerism/engraftment results are reported as the per-
cells are collected and lysed for DNA isolation. It
centage of recipient cells and/or percentage of donor
is not necessary to detach the cells from the beads
cells in the bone marrow, blood, or cell fraction. These
for most procedures.
results do not reflect the absolute cell number, which
Automated cell sorter systems may also be
could change independently of the donor/recipient ratio.
used for this purpose. With a positive selection
Inability to detect donor or recipient cells does not mean
program, for example, the instrument is capable of
that the cell population is completely absent because
isolating up to 2 × 108 pure T cells per separation.
capillary electrophoresis and fluorescent detection
Unwanted cells can be removed with the depletion
methods offer a sensitivity of 0.1% to 1% for autosomal
programs.
STR markers. Time trends may be more important than
single-point results following transplantation.
Because cell lineages engraft with different kinetics,
Advanced Concepts testing of blood and bone marrow may yield different
levels of chimerism. Bone marrow will contain more
Positive or negative selection techniques may be myeloid cells, and blood will contain more lymphoid
used to test specific cell lineages. For example, cells. The first determination to be made from engraft-
analysis of the T-cell fraction separately is used to ment testing is whether donor engraftment has occurred
and, secondly, whether there is split chimerism. In split
chimerism, cell separation techniques may be used to 120 140 160 180 200 220 240 260 280 300 320
determine which lineages are mixed and which are fully Reference
donor.
QUALITY ASSURANCE FOR SURGICAL D5S818 D13S317 D7S820 D16S539

SECTIONS USING STR
Test
The molecular diagnostics laboratory can assist in ensur-
ing that surgical tissue sections are properly identified
and not contaminated. During processing of tissue spec-
imens, microscopic fragments of tissue may persist in D5S818 D13S317 D7S820 D16S539
paraffin baths (floaters).37 These fragments can adhere
to subsequent tissue sections, resulting in the anomalous Reference
appearance of the tissue under the microscope. If a tissue
sample is questioned, STR identification can confirm the
origin of tissue.38,39,45,46
For this procedure, the suspect tissue must be care- vWA TH01 Amelo TP0X CSF1P0
fully removed from the slide by microdissection. Ref-
erence DNA isolated from the patient and DNA isolated Test
from the tissue in question are subjected to multiplex
PCR. The results are compared for matching alleles. If
the tissue in question originated from the patient, all
alleles should match. Assuming good-quality data, one vWA TH01 Amelo TP0X CSF1P0
nonmatching locus excludes the tissue in question as
coming from the reference patient. FIGURE 10.24 Quality assurance testing of a tissue frag-
An example of such a case is shown in Figure 10.24. ment. The STR profile of the fragment in question (test) was
A uterine polyp was removed from a patient for micro- compared with that of reference DNA from the patient. The
scopic examination. An area of malignant tissue was alleles matched at all loci, supporting the genotypic identity of
present on the slide. The pathologists were suspicious the test material with the patient.
about the malignancy because there was no other malig-
nant tissue observed in other tissue from the patient. The source. A second biopsy, however, also contained malig-
tissue fragment was microdissected from the thin section nant cells similar to those found in the first biopsy. The
and tested at nine STR loci. The allelic profile was com- allele differences were determined to be a result of
pared to reference DNA from the patient. The profiles microsatellite instability in the malignant cells. Another
were identical, confirming that the tissue fragment was study comparing STR alleles in genetically stable and
from the patient. unstable tumors directly demonstrated the presence of
Using STR for this application has some limitations. new alleles in unstable tumor tissue compared to normal
Tissue quality and quantity can adversely affect ampli- reference tissue from the same patient.41 It is advisable,
fication of the STR loci, especially the larger products. therefore, to take into account whether tumor cells might
Also, DNA isolated from small fragments may be mixed be genetically unstable when testing for contaminants.
with reference DNA, complicating interpretation and
comparison of alleles. A reported case study also demon-
strated the effect of inherent genomic instability in tumor SINGLE-NUCLEOTIDE POLYMORPHISMS
tissue.40 Allele differences between the suspected floater
of malignant tissue and reference tissue from the patient Data from the Human Genome Project revealed that the
led to the initial conclusion that it was from another human nucleotide sequence differs every 1,000 to 1,500
bases from one individual to another.42 The majority of

these sequence differences are variations of single nucle- TABLE 10.9 Types of SNPs
otides, or SNPs. The traditional definition of polymor-
phism requires that the genetic variation be present at SNP Region Alteration
a frequency of at least 1% of the population. The Inter- Type I Coding Nonconservative
national SNP Map Working Group observed that two
haploid genomes differ at 1 nucleotide per 1,331 bp.43 Type II Coding Conservative
This rate, along with the theory of neutral changes Type III Coding Silent
expected in the human population, predicts 11 million
sites in a genome of 3 billion bp that vary in at least 1% Type IV Noncoding 5’ UTR*
of the world’s population. In other words, each individ-
Type V Noncoding 3’ UTR
ual has 11 million SNPs. Higher-density analysis made
possible by next-generation sequencing has determined Type VI Noncoding, other
that SNPs are even more frequent—one SNP per every
300 nucleotides in a given human genome.44 *Untranslated region.
Due to the density of SNPs across the human

genome, these polymorphisms were of great interest for
genetic mapping, disease prediction, and human identi-
fication. The problem was that detection of single-base- Haplotype
pair changes was not as easy as the detection of STRs, ~10,000 bp
VNTRs, or even RFLPs.
With improving technology, mapping studies
achieved denser coverage of the genome.45 The most
definitive way to detect SNPs has been by direct
sequencing. A number of additional methods were
subsequently designed to detect known SNPs. Next-
generation sequencing has greatly accelerated both the
discovery and detection of SNPs.46,47 Computer analysis
is also required to confirm that the population frequency
of the SNPs meets the requirements of a polymorphism.
So far, approximately 10 million SNPs have been iden- FIGURE 10.25 Sections of DNA along chromosomes can be
tified in the human genome. Almost all (99%) of these inherited as a unit or block of sequence in which no recombina-
have no biological effect. Over 60,000, however, are tion occurs. All the SNPs on that block comprise a haplotype.
within genes, and some are associated with disease. A
familiar example is the SNP responsible for the forma-
The Human Haplotype Mapping
tion of hemoglobin S in sickle cell anemia. SNPs have
(HapMap) Project
been classified according to location, relation to coding
sequences, and whether they cause a conservative or The international HapMap Project was an initiative
nonconservative sequence alteration (Table 10.9). started as the Human Genome Project neared com-
SNP databases such as dbSNP, dbVar, ClinVar, and pletion. Its goal was to develop a haplotype map of
others are collections of DNA sequence variants used as the human genome (identify blocks of DNA polymor-
a reference for screening genomic sequencing data. A phisms that are inherited together; Fig. 10.25). This map
variant detected by sequencing may already be described would then be used to identify common disease associ-
or associated with a disease phenotype as noted in these ations and patterns of human DNA sequence variation.
databases. In addition to SNPs, these databases include Although millions of SNPs, RFLPs, VNTRs, and STRs
short deletions, insertions, and duplications that involve were discovered through the HapMap project, advances
more than one nucleotide. in genomic sequencing technology and development
of comprehensive population-based databases, such as HV 1 HV 2

the 1000 Genomes Project, has supplanted its contribu- (342 bp) (268 bp)
tions to research. In 2016, the National Center for Bio- PH1
technology Information retired the HapMap resource.
PH2
Researchers were directed to the 1000 Genomes Project PL
for population genetic and genomic information.
Mitochondrial genome
Histooricaal Higghlligghtts (16,569 bp)
The Human Haplotype Mapping project was ini-

tiated in October 2002, with a target completion
date of September 2005.48 An initial draft of the FIGURE 10.26 The mitochondrial genome is circular. The
HapMap was completed before the deadline date, hypervariable (HV) areas in the control region are shown.
and a second phase was started to generate an Mitochondrial genes are transcribed bidirectionally starting at
even more detailed map. The new phase increased promoters (PL, PH1, and PH2).
the density of SNP identification fivefold from 1
SNP per 3,000 bases to 1 SNP per 1,000 bases,
1 to 17, are generated by cleavage of the polycistronic
or a total of 3.1 million SNPs, approximately
(multiple-gene) transcript at the location of the tRNA
25% of the estimated 11 million SNPs in the
genes.
human genome.49 The improved detail of the sec-
Genes encoded on the mtDNA include 22 tRNA
ond-phase project (phase II HapMap) advanced
genes, 2 ribosomal RNA genes, and 12 genes coding for
efforts to locate specific genes involved in
components of the oxidation-phosphorylation system.
complex genetic disorders and provided insight
Mutations in these genes are responsible for neuropa-
into ancestry, recombination, and linkage disequi-
thies and myopathies. In addition to coding sequences,
librium studies.
the mitochondrial genome has two noncoding regions
that vary in DNA sequence, the hypervariable region
1 and the hypervariable region 2, or HV1 and HV2
MITOCHONDRIAL DNA POLYMORPHISMS (see Fig. 10.27). The reference mtDNA hypervariable
region is the sequence published initially by Anderson,
Mitochondria contain a circular genome of 16,569 bp. called the Cambridge reference sequence, the Oxford
The two strands of the circular mitochondrial DNA sequence, or the Anderson reference.50 Polymorphisms
(mtDNA) chromosome have an asymmetric distribution are denoted as variations from the reference sequence.
of Gs and Cs generating a G-rich heavy (H) and a C-rich Nucleotide sequencing of the mtDNA control region
light (L) strand. Each strand is transcribed from a control has been validated for the genetic characterization of
region starting at one predominant promoter, PL on the forensic specimens and disease states and for genealogy
L strand and PH on the H strand, located in sequences of studies.
the mitochondrial circle called the displacement (D)-loop In contrast to nuclear DNA, including the Y chro-
(Fig. 10.25). The D-loop forms a triple-stranded region mosome, mtDNA follows maternal clonal inheri-
with a short piece of H-strand DNA, the 7S DNA, syn- tance patterns. With few exceptions,51,52 mtDNA types
thesized from the H strand. Bidirectional transcription (sequences) are inherited maternally. These characteris-
starts from PL on the L-strand and PH1 and PH2 on the tics make possible the collection of reference material
H-strand. RNA synthesis proceeds around the circle for forensic analysis, even in cases in which generations
in both directions. A bidirectional attenuator sequence are skipped. For forensic purposes, the quality of a match
limits L-strand synthesis and, in doing so, maintains between two mtDNA sources is determined by counting
a high ratio of rRNA to mRNA transcripts from the the number of times the mtDNA profile occurs in data
H-strand (see Fig. 10.26). Mature mitochondrial RNAs, collections of unrelated individuals, so the estimate of
FIGURE 10.27 A peptide spectrum

reflects the underlying DNA sequence
variants in the form of variant amino
acids. Peak patterns (profiles) are com-
pared to reference databases. The prob-
ability of a proteomic profile being
shared by unrelated individuals is com-
puted from the size of the population
contributing spectra to the database.
m/z = mass/charge
the uniqueness of a particular mtDNA type depends on not contained within the forensic data and can be used
the size of the reference database. The more mitochon- for investigative purposes.
drial DNA sequences are entered into the database, the As all maternal relatives share mitochondrial
more powerful the identification by mitochondrial DNA sequences, the mtDNA of sisters and brothers or mothers
will become.53 and daughters will exactly match in the hypervariable
Mitochondrial nucleotide sequence data are divided region in the absence of mutations. Therefore, the use
into two components, forensic and public. The forensic of mtDNA polymorphisms is for exclusion. There is an
component consists of anonymous population profiles average of 8.5 nucleotide differences between mtDNA
and is used to assess the extent of certainty of mtDNA sequences of unrelated individuals in the hypervariable
identifications in forensic casework. All forensic pro- region.
files include, at a minimum, a sequence region in HV1
(nucleotide positions 16024 to 16383) and a sequence
region in HV2 (nucleotide positions 53 to 372). These
data are searched through the CODIS program in open Advanced Concepts
case files and missing persons cases. Approximately
610 bp, including the hypervariable regions of mtDNA, In contrast to nuclear DNA, the human mtDNA
are routinely sequenced for forensic analysis. Deviations genome is completely sequenced and numbered.
from the Cambridge reference sequence are recorded as Variants in the mitochondrial DNA are indicated in
the number of the position and a base designation. For relation to the full mitochondrial DNA sequence.
example, a transition from A to G at position 263 would Descriptions are preceded by “m.” and reported
be recorded as 263 G. with the terms used for nuclear DNA (e.g., a T to
The public data consist of mtDNA sequence data C change at position 8993 would be m.8993T>C).
from the scientific literature and the GenBank and Euro- Descriptions of changes at the protein level include
pean Molecular Biology Laboratory databases. The a reference to the protein changed; for example,
public data have not been subjected to the same quality replacement of leucine with proline at position 156
standards as the forensic data.54 The public databases in ATP synthase 6 would be ATP6:p.Leu156Pro.
provide information on worldwide population groups
The Scientific Working Group for DNA Methods concordance, then the test specimen cannot be excluded
(SWGDAM) and the International Society for Forensic as coming from the same individual or maternal rela-
Genetics (ISFG) have recommended guidelines for the tive as the source of the reference sequence. The conclu-
use of mtDNA from bone, teeth, or hair for identification sion that an individual can or cannot be eliminated as a
purposes.55,56 The process begins with a visual inspection possible source of mtDNA is reached under conditions
of the specimen. Bone or teeth specimens are examined defined by each individual laboratory. In addition, evalu-
and ascertained to be of human origin. In the case of ation of cases in which heteroplasmy may have occurred
hair samples, the hairs are examined microscopically and is laboratory-defined.
compared with hairs from a known source. Sequencing The mtDNA profile of a test sample can also be
is performed only if the specimen meets the criteria searched in a population database. Population databases,
of origin and visual matching to the reference source. such as the mtDNA population database and CODIS, are
Before DNA isolation, the specimens are cleaned with used to assess the weight of forensic evidence, based
detergent or, for bone or teeth, by sanding to remove on the number of different mitochondrial sequences
any possible source of extraneous DNA adhering to the previously identified. The quality of sequence informa-
specimen. The cleaned specimen is then ground in an tion used and submitted for this purpose is extremely
extraction solution. Hair shafts yield mtDNA, as does important.58,59 Based on the number of known mtDNA
the fleshy pulp of teeth or bone. The dentin layer of old sequences, the probability of sequence concordance in
tooth samples will also yield mtDNA. DNA is isolated two unrelated individuals is estimated at 0.003. The
by organic extraction and amplified by PCR. The PCR probability that two unrelated individuals will differ by
products are then purified and subjected to dideoxy a single base is 0.014.
sequencing. A positive control of a known mitochondrial Mitochondrial DNA analysis is also used for lineage
sequence is included with every run along with a reagent studies and to track population migrations. As with the
blank for PCR contamination and a negative control for Y chromosome, there is no recombination between mito-
contamination during the sequencing reaction. If the neg- chondria, and polymorphisms arise mostly through muta-
ative or reagent blank controls yield sequences similar tion. The location and divergence of specific sequences
to the specimen sequence, the results are rejected. Both in the HV regions of mitochondria are a historical record
strands of the specimen PCR product must be sequenced. of the relatedness of populations.
Raw mitochondrial sequence data are imported into Because mitochondria are naturally amplified
a software program for analysis. With the sequence (hundreds per cell and tens of circular genomes per
software, the heavy-strand sequences should be reverse- mitochondria) and because of the nuclease- and
complemented so that the bases are aligned in the damage-resistant circular nature of the mitochondrial
light-strand orientation for strand comparison and base DNA, mtDNA typing has been a useful complement to
designation. other types of DNA identification. Challenging speci-
Occasionally, more than one mtDNA population mens of insufficient quantity or quality for nuclear DNA
is present in the same individual. This is called het- analysis may still yield useful information from mtDNA.
eroplasmy. In point heteroplasmy, two DNA bases To this end, mtDNA analysis has been helpful for the
are observed at the same nucleotide position. Length identification of missing persons in mass disasters or for
heteroplasmy is typically a variation in the number of typing ancient specimens.60 MtDNA typing can also be
bases in tracts of like bases (homopolymeric tracts, applied to quality assurance issues, as described for STR
e.g., CCCCC). A length variant alone cannot be used to typing of pathology specimens.61
support an interpretation of exclusion.57
In general, if two or more nucleotide differences occur
between a reference and a test sample, the test sample
OTHER IDENTIFICATION METHODS
can be excluded as originating from the reference or a
Protein-Based Identification
maternally related person. One nucleotide difference
between the samples is interpreted as an inconclusive Like DNA, protein contains polymorphisms. Protein
result. If the test and reference samples show sequence polymorphisms are in the form of amino acid sequence
variations. Some proteins are chemically more stable Epigenetic Profiles

than DNA in harsh environments. Proteins, such as
keratin and collagen, are also more abundant. It has DNA and probably protein identification systems cannot
been proposed that protein polymorphisms may serve as distinguish between syngeneic individuals (identical
supportive confirmation or even an alternative for DNA twins). Epigenetic changes occur as a result of environ-
identification results.62 mental events, such that a putative epigenetic profile is
Nonsynonymous DNA polymorphisms produce unique to each individual because no two individuals
single-amino-acid polymorphisms in proteins. A hair will have the same environmental exposures.
shaft, for example, contains over 300 nuclear and mito- Epigenetic alterations, particularly DNA methyla-
chondrial proteins, adequately representing the whole tion, change in the absence of cell division or DNA
genome. A collection of peptide variants comprises a sequence alterations. Many of these changes are stable
profile. Like STR profiles, peptide profiles could be col- and can be detected at the DNA sequence level. There
lected for identification and population-based studies. are a variety of methods to detect methylated DNA,
By associating peptide changes with the known SNP including methylation-sensitive restriction enzymes,
that codes for them, a putative DNA profile might be methylation-specific PCR, and bisulfate sequencing by
generated from peptide data where sufficient DNA was Sanger or massive parallel sequencing.
not available. Although shared epigenetics in families is evidence
Peptide variants in these proteins can be identi- for the inheritance of epigenetic traits, epigenetic differ-
fied using liquid chromatography followed by mass ences due to environmental exposures add an additional
spectrometry. Proteins isolated from test samples are level of distinction among individuals. Epigenetic dif-
reduced, alkylated, and digested with trypsin, and the ferences, not present at early ages, have been observed
resulting peptides are resolved by liquid chromatog- in adult identical twins.65,66 Estimation of the age of
raphy. On-column concentration can increase target persons whose biological materials are recovered at a
molecule concentrations. Particles are then deposited crime scene is valuable in forensic applications. Strate-
on the matrix, ionized, and subjected to separation by gies have been designed to use epigenetic patterns that
size and charge to generate a spectrum (Fig. 10.27) accumulate over time to predict chronological age.67
that can be compared with peptide reference spectra. Through epigenetics, materials commonly found at
Software matching algorithms identify peptide variants crime scenes, such as bloodstains, may provide useful
from the reference spectra.63 A set of variants is the pro- information on human age.68
teomic profile. This profile can then be compared to a Epigenetic markers can also be used to identify body
peptide database.64 There are close to 4 million spectra fluids (e.g., saliva, semen, blood). Unique methylation
in available libraries, accounting for different ionization patterns occur at specific gene promoters in these cell
methods. Based on the population frequency of variants types.69 A marker set defined for fluid type discrimina-
(or their associated SNP alleles), the probability of a tion may also be used to eliminate fluids from other non-
proteomic profile being shared by unrelated individuals primate species.70
is computed from the size of the population contributing
spectra.
Annotated reference peptide libraries from various Case Study 10.1
organisms and proteins are being developed for the rapid
matching and identification of acquired peptide spectra A 32-year-old woman was treated for mantle cell
from non-human species as well. Among these, smaller, lymphoma with a non-myeloablative bone marrow
more focused libraries have been collected specifically transplant. Before the transplant and after a donor
for humans and mice. The National Institute of Stan- was selected, STR analysis was performed on the
dards and Technology is also developing a peptide mass donor and the recipient to find informative alleles.
spectral library to provide peptide reference data for One hundred days after the transplant, engraftment
disease-related spectra.
was evaluated using the informative STR alleles. Treatment with a tyrosine kinase inhibitor and a
The results from one marker, D5S818, are shown bone marrow transplant were recommended. The
in the top panel. One year later, the patient was man had a twin brother, who volunteered to donate
reevaluated. The results from the same marker are bone marrow. The two brothers were not sure if
shown in the bottom panel. they were fraternal or identical twins. Donor and
recipient buccal cells were sent to the molecular
120 125 130 135 140 120 125 130 135 140 pathology laboratory for STR informative analy-
REC-PRE REC-PRE sis. The results are shown here.
120 140 160 180 200 220 240 260 280 300
41919 41919 M.K.
DON-PRE DON-PRE
D5S818 D13S317 D7S820 D16S539
61188 61188 R.K.
POST POST
D5S818 D13S317 D7S820 D16S539
40704 3171 53400

M.K.
Results from engraftment analysis at 100 days (top) and 1 year
(bottom) showing marker D5S818. REC, recipient; DON,
donor. vWA TH01 TP0X CSF
QUESTION: Was the woman successfully engraft- R.K.

ed with donor cells? Explain your answer.
vWA TH01 TP0X CSF
M.K.
LPL F13B FESFPS F13A

Case Study 10.2
R.K.
A 26-year-old young man reported to his doctor
with joint pain and fatigue. Complete blood count
and differential counts were indicative of chronic LPL F13B FESFPS F13A
myelogenous leukemia. The diagnosis was con-
firmed by karyotyping, showing 9/20 metaphases STR analysis of two brothers, one who serves as bone marrow
with the t(9;22) translocation. Quantitative PCR donor (D) to the other (R). Twelve loci are shown.
was performed to establish a baseline for moni- QUESTION: Were the brothers fraternal or identi-
toring tumor load during and following treatment. cal twins? Explain your answer.
Case Study 10.3 STUDY QUESTIONS

The pathology department received a fixed par-
1. Consider the following STR analysis.
affin-embedded tissue section with a diagnosis
of benign uterine fibroids. Slides were prepared
for microscopic study. Only benign fibroid cells Locus Child Mother AF1 AF2
were observed on all slides, except one. A small
malignant process was observed located between D3S1358 15/15 15 15 15/16
the fibroid and normal areas on one slide. Because vWA 17/18 17 17/18 18
similar tissue was not observed on any other
section, it was possible that the process was a con- FGA 23/24 22/23 20 24
tamination from the embedding process. To deter- TH01 6/10 6/7 6/7 9/10
mine the origin of the malignant cells, DNA was
extracted from the malignant area and compared TPOX 11/11 9/11 9/11 10/11
with DNA extracted from normal tissue from the CSF1PO 12/12 11/12 11/13 11/12
patient. The results are shown here.
D5S818 10/12 10 11/12 12
120 140 160 180 200 220 240 260 280 300 320
D13S317 9/10 10/11 10/11 9/11
P
a. Circle the child’s alleles that are inherited from

the father.
T b. Which alleged father (AF) is not excluded as
the biological parent?
P 2. The following evidence was collected for a

criminal investigation.
T Locus Victim Evidence Suspect
TPOX 11/12 12, 11/12 11
CSF1PO 10 10, 9 9/10

STR analysis of suspicious tissue discovered on a paraffin
section. Eight loci were tested. P, patient; T, tissue section. D13S317 8/10 10, 8/10 9/12
QUESTION: Were the malignant cells seen in one D5S818 9/11 10/11, 9/11 11
section derived from the patient, or were they a
contaminant of the embedding process? Explain TH01 6/10 6/10, 8/10 5/11
your answer. FGA 20 20, 20/22 20
vWA 15/17 18, 15/17 15/18
D3S1358 14 15/17, 14 11/12
The suspect is heterozygous at the amelogenin

locus.
a. Is the suspect male or female?

b. In the evidence column, circle the alleles Locus Donor Alleles Recipient Alleles
belonging to the victim. FESFPS 10 7
c. Should the suspect be held or released?
F13A01 5,11 5,11
3. A child and an alleged father (AF) share alleles
with the following paternity index. Which loci are informative?
Paternity Index 6. An engraftment analysis was performed by

Locus Child AF for Shared Allele capillary gel electrophoresis and fluorescence
D5S818 9,10 9 0.853 detection. The fluorescence as measured by the
instrument under the FESFPS donor peak was
D8S1179 11 11,12 2.718 28,118 units, and that under the FESFPS recipient
D16S539 13,14 10,14 1.782
peak was 72,691. What is the percent donor in this
specimen?
a. What is the combined paternity index from 7. The T-cell fraction from the blood sample in
these three loci? question 6 was separated and measured for donor
b. With 50% prior odds, what is the probability of cells. Analysis of the FESFPS locus in the T-cell
paternity from these three loci? fraction yielded 15,362 fluorescence units under
the donor peak and 97,885 under the recipient
4. Consider the following theoretical allele peak. What does this result predict with regard to
frequencies for the loci indicated. T-cell-mediated events such as graft-versus-host
disease or graft-versus-tumor?
Locus Alleles Allele Frequency
8. If a child had a Y haplotype including DYS393
CSF1PO 14 0.332 allele 12, DYS439 allele 11, DYS445 allele 8, and
D13S317 9,10 0.210, 0.595 DYS447 allele 22, what are the predicted Y alleles
for these loci of the natural father?
TPOX 8,11 0.489, 0.237
9. Which of these would be used for a surname
a. What is the overall allele frequency for this test: Y-STR, MINI-STR, mitochondrial typing, or
genotype, using the product rule? autosomal STR?
b. What is the probability that this DNA found at
two sources came from the same person? 10. An ancient bone fragment was found and said
to belong to an ancestor of a famous family.
5. STR at several loci were screened by capillary Living members of the family donated DNA
electrophoresis and fluorescent detection for for confirmation of the relationship. What
informative peaks prior to a bone marrow type of analysis would likely be used for
transplant. The following results were observed. this test? Why?
Locus Donor Alleles Recipient Alleles 11. What is a biological exception to positive
identification by autosomal STR?
LPL 7,10 7,9
12. A partial STR profile was produced from a highly
F13B 8,14 8
degraded sample. Alleles matched to a reference
sample at five loci. Is this sufficient for a legal 12. Marjanović D, Durmić-Pasić A, Kovacević L, Avdić J,
identification? Dzehverović M, Haverić S, Ramić J, Kalamujić B, Lukić Bilela
L, Skaro V, Projić P, Bajrović K, Drobnic K, Davoren J, Primorac
D. Identification of skeletal remains of Communist Armed Forces
13. What is an SNP haplotype? What are tag SNPs? victims during and after World War II: Combined Y-chromosome
(STR) and MiniSTR approach. Croation Medical Journal 2009;
14. Which of the following is an example of linkage 50:296–304.
disequilibrium? 13. Kayser M, Caglià A, Corach D, Fretwell N, Gehrig C,
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Chapter 11
Detection and Identification
of Microorganisms
Outline Restriction Fragment Length Polymorphism Analysis

Arbitrarily Primed PCR
SPECIMEN COLLECTION Amplified Fragment Length Polymorphism (AFLP) Assay
SAMPLE PREPARATION Interspersed Repetitive Elements
QUALITY ASSURANCE Internal Transcribed Spacer Elements
Controls spa Typing
Quality Control Multilocus Sequence Typing
Selection of Sequence Targets for Detection of Microorganisms Mass Spectrometry
MOLECULAR DETECTION OF MICROORGANISMS Comparison of Typing Methods
Bacteria
Respiratory Tract Pathogens
Urogenital Tract Pathogens
Viruses Objectives
Mass Spectrometry
Mycology 11.1 Name the organisms that are common targets for
Parasites molecular-based laboratory tests.
ANTIMICROBIAL AGENTS 11.2 Identify advantages and disadvantages of using
Resistance to Antimicrobial Agents molecular-based methods as compared with
Molecular Detection of Resistance traditional culture-based methods in the detection
Beta-Lactam Antibiotic Resistance and identification of microorganisms.
Glycopeptide Antibiotic Resistance 11.3 Differentiate between organisms for which
Antimicrobial Resistance in M. tuberculosis commercially available nucleic acid amplification
MOLECULAR EPIDEMIOLOGY tests exist and those for which “home-brew”
Molecular Strain Typing Methods for Epidemiological Studies polymerase chain reaction (PCR) is used.
Plasmid Analysis 11.4 List the genes involved in the emergence of
Pulsed-Field Gel Electrophoresis antimicrobial resistance that can be detected by
nucleic acid amplification methods.
301
11.5 Compare and contrast the molecular methods Additionally, molecular-based tests have been devel-
that are used to type bacterial strains in oped for organisms that are received in clinical laborato-
epidemiological investigations. ries in high volumes, such as Streptococcus pyogenes in
11.6 Explain the value of controls, in particular, throat swabs and Neisseria gonorrhoeae and Chlamydia
amplification controls, in ensuring the reliability of trachomatis in genital specimens. Furthermore, genes
PCR results. that confer resistance to antimicrobial agents are the
11.7 Interpret pulsed-field gel electrophoresis patterns targets of molecular-based methodologies, such as mecA,
to determine whether two isolates are related to which contributes to the resistance of Staphylococcus
or different from each other. aureus to oxacillin; vanA, vanB, and vanC, which give
Enterococcus resistance to vancomycin; tonB, which
confers resistance to carbapenems; and katG and inhA,
which mediate M. tuberculosis resistance to isoniazid.
Furthermore, mass spectrometry can directly identify
Microbiological applications for the clinical laboratory resistance factors such as expressed β-lactamases even
are increasingly based on the molecular characterization in the absence of antibiotics.1
of microorganisms and the development and evaluation Finally, characterization of DNA, RNA, and protein
of molecular-based laboratory tests of clinical specimens was developed to find and identify new organisms and
isolated in cultures. Another important application of to further characterize or classify known organisms,
molecular technology in the clinical microbiology lab- such as influenza virus.2 Nucleic acid sequence infor-
oratory is in the comparison of biochemically similar mation is used to reclassify bacterial organisms based
organisms in outbreak situations, known as molecular on 16S rRNA sequence homology, for epidemiological
epidemiology, to ascertain whether the isolates have a purposes, and to predict therapeutic efficacy. Mass spec-
common or independent source. Clinically important trometry is also being applied to the identification of
microorganisms include a range of life-forms, from microorganisms based on peptide profiles.
arthropods to prions, and although molecular-based The molecular methods used in the clinical micro-
methods have become routine in clinical microbiol- biology laboratory are the same as those that were
ogy, traditional culture and biochemical testing are still described previously for the identification of human
important for the detection and identification of a variety polymorphisms and those that will be discussed in sub-
of microorganisms. sequent chapters for the identification of genes involved
In contrast to the analysis of phenotypic traits (micro- in cancer and in inherited diseases. These include poly-
scopic and colonial morphologies, enzyme or pigment merase chain reaction (PCR)—traditional, real-time,
production, carbohydrate fermentation patterns), the and reverse transcriptase PCR, and DNA sequencing.
analyte for molecular testing is the genome, transcrip- Additional methods used in molecular epidemiology are
tome, or proteome of the microorganism. Bacteria, fungi, pulsed-field gel electrophoresis (PFGE), matrix-assisted
and parasites have DNA genomes, whereas viruses can laser desorption ionization (MALDI) spectrometry, and
have DNA or RNA genomes. Prions, which cause trans- other methods that are discussed in this chapter. The
missible encephalopathies such as Creutzfeldt–Jakob development of molecular-based methods has been suc-
disease, consist only of protein. cessful for some organisms but not yet for all organisms,
Microorganisms targeted by molecular-based lab- as discussed in this chapter.
oratory tests have been those that are difficult and/
or time-consuming to isolate, such as Mycobacterium
tuberculosis as well as other species of Mycobacterium; SPECIMEN COLLECTION
those that are hazardous with which to work in the
clinical laboratory, such as Histoplasma and Coccidioi- As with any clinical test, proper procedure is impor-
des; and those for which reliable laboratory tests were tant for collection and transport of specimens for infec-
lacking, such as hepatitis C virus (HCV) and human tious disease testing. Microbiological specimens may
immunodeficiency virus (HIV). require special handling to preserve the viability of the
Chapter 11 • Detection and Identification of Microorganisms 303
target organism. Special collection systems have been

designed for the collection of strict anaerobes, viruses, TABLE 11.1 Specimen Transport Systems
and other fastidious organisms. Although viability is
not as critical for most molecular testing, the quality Type Examples
of nucleic acids may be compromised if the specimen Sterile Sterile cups, screw-capped tubes,
is improperly handled. DNA and especially RNA will containers stoppered tubes, Petri dishes
be damaged in lysed or nonviable cells. Due to the
sensitivity of molecular testing, it is also important to Swabs Calcium alginate swabs, Dacron
swabs, cotton swabs, nasopharyngeal-
avoid contamination that could yield false-positive urogenital swabs, swab transport system
results.
Collection techniques designed to avoid contami- Specialty Neisseria gonorrhoeae transport systems,
nation from the surrounding environment of adjacent systems Swab Extraction Tube System (SETS)
tissues apply to molecular testing, especially to those Proprietary Molecular testing, N. gonorrhoeae
tests that use amplification methods. Sampling must systems transport systems, STAR buffer42
include material from the original infection. The time
Anaerobic Starplex Anaerobic Transport system
and site of collection should be optimal for the likely
transport (Fisher), BBL Vacutainer Anaerobic
presence of the infectious agent. For example, Salmo- systems Specimen Collector
nella typhi is initially present in peripheral blood but not
in urine or stool until at least 2 weeks after infection. For Viral transport BD Cellmatics Viral Transport Pack, BBL
systems Viral Culturette
classical methods that include culturing of the agent, a
sufficient number of microorganisms must be obtained
for agar or liquid culture growth. For molecular testing,
however, minimum numbers (as few as 50 organisms)
can be detected successfully. The quantity of target
organisms, as well as the clinical implications, should be
taken into account when interpreting the significance of
positive results. Molecular detection can reveal infective
agents at levels below clinical significance. Conversely,
highly specific molecular methods may miss detection
of a variant organism.
Equipment and reagents used for specimen collection
are also important for molecular testing (Table 11.1).
Blood draws should go into the proper anticoagulant,
if one is to be used. Although wooden-shafted swabs
may be used for throat cultures, Dacron or calcium algi-
FIGURE 11.1 The Swab Extraction Tube System (SETS)
nate swabs with plastic shafts have been recommended
consists of a punctured inner tube that holds a swab and fits in
for collection of bacteria, viruses, and mycoplasma a capable outer tube. Under the force of centrifugation, liquid
from mucosal surfaces.3 The plastics are less adherent in the swab is forced through the inner tube and into the outer
to the microorganisms and will not interfere with PCR tube, where it can be stored.
reagents, with the exception of calcium alginate swabs
with aluminum shafts, which had been reported to affect
PCR amplification.4 Collection media or amplifica- Commercial testing kits supply an optimized collec-
tion system may influence positivity rates.5 Collection tion system for a particular test organism. The Clinical
methods such as Swab Extraction Tube System (SETS), and Laboratory Standards Institute has published docu-
sonication, and vortex have been designed for maximum ments addressing the requirements for transport devices
recovery of microorganisms from swabs by centrifuga- and quality control guidelines. The College of American
tion6 (Fig. 11.1). Pathologists requires documented procedures describing
specimen handling, collection, and transport in each preparation methods. Finally, if RNA is to be analyzed,
laboratory. inactivation or removal of RNases in the sample and in
all reagents and materials that come into contact with the
sample is important.
SAMPLE PREPARATION Any clinical specimen can be used as a source of
microorganism nucleic acid for analysis. Depending on
Isolating nucleic acids from microorganisms is similar the specimen, however, special preparation procedures
to isolating nucleic acids from human cells with only may be necessary to allow for optimal nucleic acid
a few additional considerations. First, depending on the isolation, amplification, and analysis. The presence of
microorganism, more rigorous lysis procedures may be inhibitors of DNA polymerase has been demonstrated in
required. Mycobacteria and fungi, in particular, have clinical samples; therefore, careful separation of nucleic
thick cell walls that are more difficult to lyse than those acid from other molecules present in the sample will
of other bacteria and parasites. Gram-positive bacteria ensure target amplification.7 When processing a whole-
has a thicker cell wall than gram-negative bacteria and blood specimen, it is important to remove hemoglobin
may require more rigorous cell lysis conditions. Myco- and other products of metabolized hemoglobin because
plasma, on the other hand, lacks a cell wall, and so care they can inhibit DNA polymerase and thus may prevent
must be taken with the sample to avoid spontaneous the amplification of nucleic acid in the sample, resulting
lysis of the cells and loss of nucleic acids. in a false-negative PCR result. Eukaryotic cells can be
used as a source of nucleic acid for organisms, primarily
viruses that infect these cells. In blood samples, white
Advanced Concepts blood cells are isolated from the red blood cells using
Ficoll-Hypaque and then lysed. Alternatively, whole
Biological safety is an important concern for clin-
blood is processed in automated DNA isolation systems,
ical microbiology. Because various collection,
which effectively remove hemoglobin and other con-
transport, and extraction systems inactivate organ-
taminating molecules. Serum and plasma (devoid of red
isms at different times, the technologist should
blood cells) are also used as sources of microorganism
follow the recommendations of the Centers for
nucleic acid.
Disease Control and Prevention (CDC) that call
Sputum is a source of nucleic acid from organisms
for universal precautions, treating all specimens as
that cause respiratory tract infections. Acidic poly-
if they were infectious throughout the extraction
saccharides present in sputum may inhibit DNA poly-
process. Updated guidelines are available from
merase and thus must be removed. Using a method or
the CDC for the handling of suspected bioterror-
an instrument that reliably separates DNA from other
ism material. Organisms such as smallpox must
cellular molecules is sufficient to remove the inhibitors.
be handled only in approved (level 4 contain-
Urine, when sent for nucleic acid isolation and amplifi-
ment) laboratories. Molecular testing has eased
cation, is treated similarly to cerebrospinal fluid; that is,
the requirements for laboratory culture. Methods
the specimen is centrifuged to concentrate the organisms
devised to replace the growth of cultures should
and then subjected to nucleic acid isolation procedures.
improve safety levels.
Inhibitors of DNA polymerase—nitrate, crystals, hemo-
globin, and beta-human chorionic gonadotropin—have
Second, the concentration of organisms within the clin- been demonstrated in urine as well.8
ical sample must be considered. Samples can be centri- The type of specimen used for molecular testing
fuged to concentrate the organisms within the fluid from will also affect extraction and yield of nucleic acid.
the milliliters of sample that are often received down to For example, viral nucleic acid from plasma is easier
volumes that are appropriate for molecular procedures. to isolate than nucleic acid from pathogenic Entero-
Third, inhibitors of enzymes used in molecular anal- coccus in stool specimens. Reagents and devices have
ysis may be present in clinical specimens; removal or been developed to combine collection and extraction of
inactivation of inhibitors might be included in specimen nucleic acid from difficult specimens; for example, stool
transport and recovery (STAR) buffer or the FTA paper a positive amplification control signal, whereas the
systems that inactivate infectious agents and adhere reagent blank should be negative for amplification. With
nucleic acids to magnetic beads or paper, respectively. a positive amplification control, lack of amplification
of the target can be more confidently interpreted as a
true negative result. Amplification controls are usually
QUALITY ASSURANCE housekeeping genes or those that are always present in
human or microbiological samples. Housekeeping genes
For any type of medical laboratory procedure, quality that are used as internal controls include prokaryotic
control is critical for ensuring the accuracy of patient genes, such as groEL, rpoB, recA, and gyrB, and eukary-
results. Ensuring the quality of the molecular methods is otic genes, such as β-actin, glyceraldehyde-3-phosphate,
equally important. Molecular testing sensitivity is rela- interferon-γ, extrinsic homologous control, human mito-
tively high, so even one molecule of target is a potential chondrial DNA, and peptidylprolyl isomerase A.9
template. Thus, the integrity of specimens, that is, spec- Internal controls are amplification controls that mon-
imens not contaminated by other organisms or with the itor particular steps of an amplification method. They
products of previous amplification procedures, is critical can be either homologous extrinsic, heterologous ex-
to avoid inaccurate results. Also, it is equally important trinsic, or heterologous intrinsic (Fig. 11.2). A homol-
to ensure that the lack of a product in an amplification ogous extrinsic control is a target-derived control with a
procedure is due to the absence of the target organism non-target-derived sequence insert. This control is added
and not the presence of inhibitors preventing the ampli- to every sample after nucleic acid extraction and before
fication of target sequences. amplification. The amplification of this control occurs
using the same primers as for the target, which is good
for ensuring that amplification occurs in the sample
Controls
but does not control for target nucleic acid degrada-
Control substances of known composition are used to tion during extraction. Heterologous extrinsic controls
monitor the reliability of the method and the input spec- are non-target-derived controls that are added to every
imen material. The incorporation of positive controls sample before nucleic acid extraction. This control will
shows that an assay system is functioning properly. A ensure that the extraction and amplification procedures
sensitivity control that is positive at the lower limit of were acceptable, but a second set of primers must also
detection demonstrates the sensitivity of qualitative be added to the reaction for the control to be amplified.
assays. Two positive controls, one at the lower limit and Using this control requires that the procedure be opti-
the other at the upper limit of detection, are run in quan- mized so that the amplification of the control does not
titative assays to test the dynamic range of the assay. interfere with the amplification of the target. Heterolo-
Reagent blank or contamination controls are critical for gous intrinsic controls are nontarget sequences naturally
monitoring reagents for contamination; the latter con- present in the sample, such as eukaryotic genes in a test
tains all of the assay reagents except target sequences and for microorganisms. In this case, human gene controls
should always be negative. For typing and other studies serve to ensure that human nucleic acid is present in the
that might include nontarget organisms, a negative tem- sample in addition to controlling for extraction and am-
plate control might also be included. This control will plification. Using this control requires that either two am-
detect the presence of the unwanted target(s), but should plification reactions be performed on the sample, one for
not react with the desired target. the control and the other for the target gene, or that the
With regard to amplification methods that are inter- amplification procedure be multiplexed, as long as there
preted by the presence or absence of product, false- is no interference with the amplification of the target.
negative results can occur due to amplification failure.
In order to rule out this type of false-negative result,
Quality Control
an amplification control aimed at a target that is always
present can be incorporated into an amplification assay. In a procedure that detects a microorganism, a positive
The negative template control sample should have result states that the organism is present in that sample,
Target
Target organism
Homologous extrinsic
Plasmid
Heterologous extrinsic
Nontarget
organism
Heterologous intrinsic
Host
FIGURE 11.2 Amplification controls and their relationships to the molecular target. Homologous extrinsic controls are a modi-
fied version of the target that maintain the target primer binding sites. The homologous intrinsic control may be smaller, larger, or
the same size as the target. Heterologous extrinsic controls are obtained from unrelated nontarget organisms and require primers
different from those of the target sequences. Heterologous intrinsic controls are similar to heterologous extrinsic controls, except
that heterologous intrinsic controls come from host sequences.
whereas a negative result indicates that the organism the laboratory. Second, amplification procedures may be
is not present (at amounts up to the detection limits of inhibited by substances present in the specimen. Hemo-
the assay). Although most false-positive test results can globin, lactoferrin, heparin and other anticoagulants,
be eliminated by preventing carryover contamination, sodium polyanethol sulfonate (anticoagulant used in
another source of false-positive test results that cannot blood culture media), and polyamines have been shown
be controlled in the laboratory is the presence of dead to inhibit nucleic acid amplification procedures.10 Atten-
or dying microorganisms in the sample of a patient tion to nucleic acid isolation procedures and ensuring
taking antimicrobial agents. In this situation, the nucleic optimal purification of nucleic acid from other compo-
acid–based tests will remain positive longer than culture nents of the specimen and extraction reagents will help
assays and thus may appear as a false positive. Repeat- minimize the presence and influence of inhibitors on the
ing the nucleic acid–based assay 3 to 6 weeks after anti- amplification reaction.11 As with all clinical tests, vali-
microbial therapy is more likely to yield a true-negative dation must be performed on new molecular-based tests
result. False-negative results may be more problematic that are brought into the laboratory. Controls must be
and arise when the target organism is present but the tested, and the sensitivity, specificity, and reproducibility
test result is negative. There are a few reasons for false- of the assay must be determined using reference mate-
negative results on a sample. First, the organism may rials.12 Proficiency testing of methods and competency
be present, but the nucleic acid was degraded during testing of personnel should be performed regularly. The
collection, transport, and/or extraction. This degradation Clinical and Laboratory Standards Institute, Associa-
can be prevented by proper specimen handling, effective tion for Molecular Pathology, and the Food and Drug
transport media, and inhibiting the activity of DNases Administration (FDA) have guidelines for molecular
and RNases that may be present in the sample and in methods in the laboratory.
Selection of Sequence Targets for Detection HIV or other retroviruses have variable sequences within
of Microorganisms the same species. Such variations may be informative in,
for instance, determining drug resistance or for epidemi-
Molecular methods are based on sequence hybridiza- ological information; however, not all types would be
tion or recognition using known nucleic acid sequences detected by a single sequence. The variable sequences
(primers or probes). These tests are limited by the choice may be included in the probe or primer areas to differen-
of target sequences for primer or probe hybridization. tiate between types. These type-specific probes/primers
The primary nucleotide sequence of many clinically are used in a confirmatory test after an initial test using
important microorganisms is available from the National probes or primers directed to a sequence shared by
Center for Biological Information (NCBI) or from pub- all types.
lished literature. The specificity of molecular methods In addition to their strain or species specificity, target
targeting these sequences depends on the primers or sequences must meet technical requirements for hybrid-
probes that must hybridize specifically to the chosen ization conditions. Primers should have similar anneal-
point in the genome of the microorganism. ing temperatures and yield amplicons of appropriate
Choosing a sequence target is critical for the specific- size. Probes must hybridize specifically under the con-
ity of a molecular test (Fig. 11.3). Many microorganisms ditions of the procedure. Sequence differences can be
share the same sequences in evolutionarily conserved distinguished using sequence-specific probes or primers.
genes. These sequences would not be used for detection Design of probe-based amplification or detec-
of specific strains as they are likely to cross-react over tion methods, includes decisions as to the length and
a range of organisms. Sequences unique to the target sequence structure of the probe, whether the probe is
organism are therefore selected. Some organisms such as DNA, RNA, or protein, and how the probe is labeled.
The source of the probe is also important, as probes
must be replenished and perform consistently over long-
term use. Probes are manufactured synthetically or bio-
A B C
logically by cloning. Synthetic oligonucleotides may be
Genome preferred for known sequences where high specificity
is required. Primer design includes the length and any
Target organism modifications of the primers and type of signal genera-
tion for quantitative PCR.
Many tests currently used in molecular microbiol-
Genome
ogy are supplied as commercially designed systems,
including prevalidated probes and/or primers. Several
Target organism
(variant) of these are FDA-approved or FDA-cleared methods
(http://www.fda.gov). Manufacturers of these commer-
Genome cial reagents specify requirements for quality assurance,
including controls and assay limitations. Each system
Other flora
must be validated in the testing laboratory on the type
of specimen used for clinical testing, including serum,
FIGURE 11.3 Selection of target sequences for a nucleic acid plasma, cerebrospinal and other body fluids, tissue, cul-
test. The genomes of three organisms—the test target, a variant tured cells, and organisms. In addition to the commercial
or different type of the test target, and another nontarget organ-
reagent sets, many professionals working in medical lab-
ism—are depicted. Sequence region A is not specific to the
oratories have developed in-house laboratory protocols
target organism and is, therefore, not an acceptable area for
probe or primer binding to detect the target. Sequences B and (laboratory-developed tests [LDTs]) for which primers
C are specific to the target. Sequence B is variable and can be are designed based on sequence information that has
used to detect and type the target, although some variants may been published; the reagents are bought separately, and
escape detection. Sequence C will detect variants of the target the procedures are developed and optimized within the
organism but cannot be used for determining the type. individual laboratory.
MOLECULAR DETECTION OF MICROORGANISMS following the amplification of the target (Fig. 11.4). Like
conventional PCR, qPCR is performed on nucleic acid
Molecular-based methods that have been used to detect extracted directly from clinical specimens, including
and identify bacteria include nucleic acid-based hybrid- viral, bacterial, and fungal pathogens.
ization and amplification procedures. Target detection is Designing a qPCR method requires selecting a target
accomplished by a variety of methods, including agarose gene unique to the specimen or specimen type for which
gel electrophoresis, amplification methods (PCR, TMA, primers and probes can be designed. The DNA-specific
loop-mediated isothermal amplification [LAMP]), dye, SYBR green, can be used in place of probes if
sequencing, immunoassays, western blots, and mass the amplicon is free of artifact, such as mis-primes or
spectrometry. primer dimers. The probe types most often used include
Real-time PCR, or quantitative PCR (qPCR), is fluorescent energy transfer hybridization or hydrolysis
used frequently for the detection of infectious agents probes.
because it provides a sensitive, safe closed-tube assay The requirement for probes in addition to primers
with quantitative information not available from conven- increases the complexity of the design process. Instru-
tional PCR or other “end-point” amplification methods. ment software and several websites offer computer
The quantitative capability of qPCR allows distinction programs that automatically design primers and probes
of subclinical levels of infection (qualitatively positive on submitted sequences. Commercial primer and probe
by conventional PCR) from higher levels with patho- sets are also available for purchase as reagent sets with
logical consequences. Furthermore, qPCR programs can optimized buffers and required reaction components. A
be designed to provide closed-tube sequence or typing variety of gene targets have been used for qPCR detec-
analysis by adding a melt-curve temperature program tion of a number of organisms. A list of examples of
0.08
Fluorescence (FZ/Back–F1)
0.07
BK
0.06
0.05
0.04
JC
0.03
0.02
0.01
0
55 60 65 70 75 80 85
Temperature (°C)
Fluorescence, d(FZ/Back–F1)dt
0.012
0.01 BK
JC
0.008 FIGURE 11.4 Melt-curve analysis of BK and JC
0.006 viruses. BK and JC are differentiated from one another
by differences in the Tm* of the probe specific for each
0.004
viral sequence. Fluorescence from double-stranded DNA
0.002 decreases with increasing temperature and DNA denatur-
0 ation to single strands (top panel). Instrument software
–0.002 will present a derivative of the fluorescence (bottom
60 62 64 66 68 70 72 74 76 78 80 panel) where the Tms (67°C to 68°C for BK and 73°C to
Temperature (°C) 74°C for JC) are observed as peaks. See Color Plate 9.
targets and probes is presented in a comprehensive Bacteria

review by Espy et al.13
Respiratory Tract Pathogens
Similar to standard PCR, useful genes for qPCR
methods include ribosomal RNA (rRNA), both 16S and Bacteria that cause respiratory tract disease are ubiqui-
23S, and housekeeping genes such as groEL, rpoB, recA, tous in the environment and are endemic (native to a
and gyrB. The 16S rRNA is a component of the small certain region or population group) even in higher socio-
subunit of the prokaryotic ribosome, and the 23S rRNA economic countries. Bacteria in the respiratory tract are
is a component of the large subunit of the prokaryotic easily transmitted by contact with infected respiratory
ribosome. Sequencing of the DNA region encoding secretions. Laboratory detection and identification of
16S rRNA (rDNA) is performed to determine the evo- these organisms by nonmolecular methods often lack
lutionary and genetic relatedness of microorganisms and sensitivity and/or are time-consuming. Because of their
has driven changes in microorganism nomenclature.14 importance in causing human disease, molecular-based
The rDNA that encodes the rRNA consists of alternat- assays that can detect and identify bacterial pathogens
ing regions of conserved sequences and sequences that directly in respiratory specimens have been developed
vary greatly from organism to organism. The conserved (see Table 11.2).
sequences encode the loops of the rRNA and can be Frequent testing targets include Bordetella, Legio-
used as a target to detect all or most bacteria. Sequences nella, Mycobacteria, Chlamydia, and Streptococcus
that have a great amount of heterogeneity encoded in species. Individual IVD and analyte-specific reagent
the stems of the rRNA can be used to detect a specific (ASR) systems have been marketed for individual testing
genus or species of bacteria. Ribosomal RNA was the from a variety of specimen sources. Multiplex tests are
original target of many bacterial molecular-based assays, also performed for screening or speciation.
but because of the instability and difficulty in analyzing Bordetella pertussis is a pathogen of the upper
RNA, current assays amplify and detect rDNA sequences respiratory tract that is the causative agent of whoop-
and proteins. ing cough. The organism is endemic worldwide and
Mass spectrometry of microbial proteins has been is transmitted via direct contact with infected respira-
applied to microbiological identification and epidemiol- tory secretions. Primer and probe ASR for B. pertus-
ogy.15,16 In MALDI technology, proteins are converted sis and Bordetella parapertussis detection by qPCR
into singly charged ions in an energy-absorbent matrix. targeting IS481 and IS1001, respectively, have been
For microbiological applications, the matrix is an available.
acidic compound such as sinapinic acid, or a-cyano-4- Legionella pneumophila is the cause of Legionnaires’
hydroxycinnamic acid (CHCA) dissolved in an organic disease, an infection of the lower respiratory tract
mixture of ethanol or methanol and a strong acid. The that was first described in men attending an American
solvents penetrate cell walls and membranes, extract- Legion convention in Philadelphia in 1976. Since their
ing the intracellular proteins. Some organisms can be first identification, Legionella species have been found
spotted directly from a single colony and covered with in water, both in the environment as well as in air condi-
matrix. Initial extraction in formic acid is required for tioners and hot water tanks in various types of buildings.
reproducible identification of gram-positive organisms Legionella species infections range from asymptomatic
and fungi.17 to fatal and are the third most common cause of com-
Peptide databases are the central determinant in munity-acquired pneumonias.18 PCR tests for Legionella
mass spec. These profiles, also called protein mass have targeted the macrophage infectivity potentiator
fingerprints, are maintained by instrument manufac- (mip) gene and 16S and 5S rRNA genes.
turers and also available as open-source options. In M. tuberculosis is an important cause of respiratory
vitro diagnostics (IVD) versions of databases of over tract infections causing significant levels of morbidity
200 profiles are used to identify species and strains. and mortality. The diagnosis of tuberculosis (TB) may
Several factors, including culture, sample preparation, take prolonged periods, during which time infections can
and instrument technology, influence the informativity spread. The genome of M. tuberculosis has 4.4 million
of profiles produced. base pairs (bp) with about 4,000 genes. The genomes of
TABLE 11.2 Typical Respiratory Tract Organisms Targeted by Molecular-Based Detection Methods73,74
Organism Specimen Source Gene Target Traditional Diagnostic Methods
Mycoplasma Bronchoalveolar lavage 16S rRNA Culture

pneumoniae 16S rDNA Serology
Species-specific protein gene
P1 adhesion gene
Chlamydophila Respiratory Cloned Pst I fragment Culture

pneumoniae Throat 16S rRNA
Atherosclerotic lesions MOMP
Legionella Deep respiratory secretions 5S rRNA mip gene Culture

Serum 16S rRNA Antigen detection
Buffy coat
Urine
Bordetella Nasopharyngeal IS 481 Culture

pertussis Adenylate cyclase gene Direct fluorescent antibody
Porin gene
Pertussis toxin promoter region
Streptococcus Blood DNA polymerase gene Culture

pneumoniae Cerebrospinal fluid plyA (pneumolysin)
Serum lytA (autolysin)
Sputum pbp2a (penicillin-binding protein)
pbp2b
pspA (pneumococcal surface protein)
Mycobacterium Sputum 16S rRNA Culture

tuberculosis Bronchoalveolar lavage
Bronchial washings
Gastric aspirates
different isolates of M. tuberculosis do not vary to any systems improved the detection rate of mycobacteria to
great extent, and most variation is due to the movement a few days, depending on the organism load.
of insertion elements rather than to point mutations.19 Nucleic acid amplification methodologies can detect
For many years, tuberculosis was detected from M. tuberculosis directly in a clinical specimen with
mycobacterial smears and culture. Whereas a fluoro- reliable sensitivity and specificity. PCR tests target-
chrome stain has increased sensitivity compared with the ing the species-specific sequences, such as IS6110 and
Kinyoun and Ziehl–Neelsen stains for detecting myco- 16S rRNA, allow detection of M. tuberculosis from
bacteria directly in clinical specimens. The sensitivity of fresh, frozen, or fixed tissue. PCR-positive samples are
smears in general for mycobacteria varies. At least 104 hybridized with genus-specific and species/complex-
organisms/mL are required in order to see mycobacteria specific probes. qPCR assays have also been developed
in a smear, and even then, not all of those smears read as for M. tuberculosis detection with primers and probes
positive. Cultures for M. tuberculosis are more sensitive targeting rRNA internal transcribed spacer (ITS) ele-
than smears and are able to detect 101 to 102 organisms/ ments in M. tuberculosis.
mL of specimen; however, they take time due to the slow Mycoplasma pneumoniae has been subjected to ampli-
in vitro growth of the organism. Liquid-based culture fication techniques and other characterizing methods
such as multilocus variable-number tandem-repeat molecular methods are so well characterized for these
(VNTR) analysis, multilocus sequence typing, and two organisms that they are used almost exclusively
matrix-assisted laser desorption ionization time-of-flight in the detection of the nucleic acid of N. gonorrhoeae
mass spectrometry (MALDI-TOF MS).20 and C. trachomatis. Other sexually transmitted bacte-
MALDI libraries of 50 to over 300 Mycobacterium ria are considered good targets for the development of
peptide spectra are offered by at least one manufacturer. molecular-based methods because traditional labora-
Due to the structure of their cell walls, mycobacteria tory methods of detection and identification for these
require special preparation for mass spectrometry testing. organisms either lack sensitivity or are time-consuming.
The bacteria are lysed by bead beating or in boiling Table 11.3 summarizes the molecular-based tests that
water (also a safety measure), extracted with ethanol, have been described for the bacteria that cause genital
dried, and resuspended in formic acid and acetonitrile tract infections.
for analysis. Several studies have shown favorable com- Cultures for N. gonorrhoeae and C. trachomatis
parison of MALDI-TOF with conventional methods. have been considered the gold standard, but nucleic
Chlamydophila pneumoniae is an obligate intracel- acid amplification assays have the advantages of
lular pathogen that causes 10% of community-acquired being rapid, and testing can be batched and automated,
pneumonias and has been implicated in atherosclerosis resulting in further savings for the laboratory. The first
and coronary artery disease. Despite the problems with molecular-based assay available for N. gonorrhoeae and
the development, implementation, and interpretation of C. trachomatis was a nonamplification-based nucleic
molecular-based assays for M. pneumoniae, L. pneu- acid hybridization method that detected the rRNA with
mophila, B. pertussis, and C. pneumoniae individually, an acridinium-labeled single-stranded DNA probe. DNA
multiplex nucleic acid amplification tests offer sensitive, probes have comparable sensitivities and specificities
specific, and rapid detection.21,22 to culture methods. Adding enhanced signal amplifica-
Streptococcus pneumoniae is a major cause of tion to the probe methods increased sensitivity. Numer-
community-acquired pneumonia and is also a common ous nucleic acid amplification assays are available that
cause of bacteremia, sepsis, otitis media, and meningi- target N. gonorrhoeae and C. trachomatis. These include
tis. Molecular-based tests targeting S. pneumoniae have target amplification assays, strand displacement ampli-
attempted to detect S. pneumoniae in various clinical fication, and transcription-mediated amplification. The
samples by targeting a variety of genes (see Table 11.2). nucleic acid amplification assays are performed on ure-
Although PCR is specific for S. pneumoniae, the clini- thral or cervical swabs, urine, and, in some cases, on
cal significance of a positive PCR assay is questionable transport vials that are used to collect cervical cells for
because a significant portion of the population (especially Papanicolaou smears with good sensitivity and spec-
children) is colonized with the organism, and PCR cannot ificity. Although molecular-based assays are the main
discern between colonization and infection.23,24 Sequenc- methods for detection of N. gonorrhoeae, C. tracho-
ing of 16S rRNA also does not allow enough discrimina- matis cultures may still be performed in conjunction
tion among alpha-hemolytic Streptococci species because with molecular-based assays in cases of suspected child
they share more than 99% sequence homology. Even abuse. Another consideration is laboratory testing to
MALDI-TOF identification S. pneumoniae is limited by confirm the cure of an infection. As with any infectious
database discrimination between closely related species. organism and molecular-based test, the nucleic acid is
Improvements in sample preparation methods, the use of detectable in a clinical sample whether the organism is
peak analysis, and updated databases have been applied dead or alive. It is recommended that a sample should
to distinguishing these species.25,26 not be taken for 3 to 4 weeks after treatment to confirm
treatment efficacy. Collection of samples and testing too
soon after treatment will result in positive results after
Urogenital Tract Pathogens
cultures on the same specimen have become negative.
Neisseria gonorrhoeae and Chlamydia trachomatis The spirochete T. pallidum subspecies pallidum is
were among the first organisms to be targeted for detec- the causative agent of syphilis, a sexually transmitted
tion in clinical specimens by molecular methods. The disease that results in the formation of a chancre at the
TABLE 11.3 Typical Genital Tract Organisms Targeted by Molecular-Based Detection Methods75
Organism Specimen Sources Traditional Diagnostic Methods Gene Target
Treponema Genital ulcers Serological (indirect and direct) TpN44.5a

pallidum Blood Direct antigen detection (dark field, TpN19
Brain tissue direct fluorescent antibody [DFA]) TpN39
Cerebrospinal fluid p01A
Amniotic fluid TpN47
Placenta 16S rRNA
Umbilical cord polA
Fetal tissue
Serum
Mycoplasma Urine Culture MgPa (adhesion gene)

genitalium Urethral rDNA gene
Vaginal
Cervical
Mycoplasma Genital tract Culture 16S rRNA

hominis Amniotic fluid
Ureaplasma Genital tract Culture 16S rRNA

urealyticum Amniotic fluid Urease gene
Haemophilus Gram stain 1.1 kb target

ducreyi Culture groEL gene
Serological Intergenic spacer between 16S and 23S rDNA
p27
16S rDNA gene
Neisseria Urine Culture omp III gene

gonorrhoeae Urethral opa gene
Cervical Cytosine DNA methyltransferase gene
Thin preparation vials cPPB gene
Site-specific recombinase gene
Chlamydia Urine Culture MOMP

trachomatis Urethral Enzyme immunoassay 16S RNA
Cervical DFA
Thin preparation vials
Conjunctiva
site of inoculation (primary syphilis). If left untreated, (rapid plasma reagin [RPR] and venereal disease
the organism disseminates through the body, damag- research laboratory [VDRL] tests) initially for the pres-
ing tissues. The patient may progress into the other ence of antibodies against cardiolipin (a normal compo-
stages of disease: secondary syphilis (disseminated nent of host membranes) and confirmed by testing for the
rash), latent syphilis (asymptomatic period), and ter- presence of antibodies against T. pallidum by enzyme
tiary syphilis (central nervous system and cardiovascular immunoassay to confirm infection. T. pallidum cannot
manifestations). be grown in vitro. Laboratories have adopted hemag-
Laboratory diagnosis of syphilis is limited to sero- glutination assays and enzyme immunoassays (EIAs)
logical testing, in which patients are typically screened to screen patients for syphilis and fluorescent antibody
absorption, particle agglutination assays, and the RPR the same time. Since this first description of the multi-
to monitor the effectiveness of treatment and to detect plex assay, other groups have used multiplex methods
reinfection.27,28 RPR and VDRL are limited in that, when to confirm the sensitivity of the assay for the targeted
reactive, they are not specific for syphilis, and the sensi- organisms and to examine the prevalence of these organ-
tivity of these tests in very early and late syphilis is low. isms in various geographic areas in different years.30,31
The serological tests that detect T. pallidum antibodies
are limited by their inability to differentiate between
Viruses
current and past infections. The RPR test, though, can
be used to detect reinfection because titers of anticardi- Evidence for virus infection has been detected by testing
olipin antibodies will decrease to nonreactive following for antibodies against the virus, by measuring the pres-
successful treatment of the organism and increase again ence or absence of viral antigens, or by detecting the
with reinfection. growth of a virus in a culture system. Although some of
Several PCR assays have been developed and tested these methods are well established for certain viruses,
for the direct detection of T. pallidum DNA in genital they all have major disadvantages.
ulcers, blood, brain tissue, cerebrospinal fluid, serum, Antibody detection is an indirect method of diag-
and other samples, with varying sensitivities. Amplifica- nosis. The host immune response has to be stimulated
tion of the T. pallidum DNA polymerase I gene (polA) by the virus to produce antibodies. In the case of an
is highly accurate when tested on genital, anogenital, immunodeficient patient, the lack of antibodies due to
or oral ulcers. PCR assays for T. pallidum that require host factors might be interpreted as a lack of the virus.
less than 1 mL of blood have accuracy comparable to Furthermore, antibodies are a retrospective indication
serology. of the infection. To interpret antibody testing with the
Mycoplasma hominis, Mycoplasma genitalium, and most confidence, paired sera should be collected, with
Ureaplasma urealyticum cause nongonococcal ure- one sample collected during the acute phase of the infec-
thritis. The mycoplasmas, as discussed previously for tion and the other collected as the patient is recovering.
M. pneumoniae, are the smallest free-living, self- The titers of antibodies are measured in both samples.
replicating organisms known. M. genitalium has the A fourfold or greater rise in titer level from the acute
smallest genome and thus was one of the first organisms sample to the convalescent sample indicates the pres-
to have its genome fully sequenced.29 M. genitalium ence of the virus during the acute stage. Detecting IgM
culture methods were labor intensive and not widely antibodies, in particular, during an acute infection is the
available. best evidence for the presence of a virus. But in patients
PCR assays were developed targeting the adhesion in the very early stages of infection, IgM titers may be
gene (MgPa) or the rDNA gene of M. genitalium. PCR below detection limits. During this “window” period,
was vital in establishing M. genitalium as an impor- the patient is infected and infectious.
tant genital tract pathogen, and laboratory-developed Antigen detection testing is available in the clinical
PCR with hybridization is used for clinical detection laboratory only for some viruses. Assays that measure
of M. genitalium as well as M. hominis and U. urea- viral antigens are available more often for respiratory
lyticum detection. Assay development was at one time syncytial virus (RSV), influenza virus, rotavirus, HSV,
hindered due to the absence of a reliable gold standard cytomegalovirus (CMV), and hepatitis B virus (HBV).
for comparison and especially in the absence of clini- Viral antigens are detected by enzyme immunoassays or
cal symptoms. Thus, the clinical significance of PCR- direct immunofluorescent assays.
positive specimens was difficult to interpret. Genital tract The classical method for detection and identifica-
specimens have been the target for the development of tion of viruses in body fluids is tissue or cell culture.
multiplex assays in which the presence of nucleic acid of Monolayers of host cells are grown in vitro, the patient’s
multiple organisms can be determined from one speci- specimen is inoculated onto the cells, and changes in
men in a single tube. The organisms causing genital tract the cells due to viral infection, called cytopathic effect,
infections often overlap in their symptoms, or infections are observed microscopically by the technologist. The
can be caused by the presence of multiple organisms at identity of the virus is confirmed using fluorescently
labeled monoclonal antibodies. Culture has been the

gold standard for many viruses, in particular adenovirus, TABLE 11.4 Genomes of Human Viruses
enteroviruses, CMV, influenza, and HSV, but it is not Double-Stranded DNA Viruses Adenovirus
applicable to other viruses, such as the hepatitis viruses, BK virus
because these viruses do not grow well in culture Cytomegalovirus
systems. Another disadvantage to viral culture is the Epstein–Barr virus
amount of time required before viral growth is detect- Hepatitis B virus
Herpes simplex virus 1
able. Although the shell vial system, where the sample Herpes simplex virus 2
is centrifuged onto a cell monolayer and tested with anti- Human papillomavirus
viral antibodies, decreased detection time, several days JC virus
to weeks, depending on the virus, can pass before detec- Molluscum virus
tion of cytopathic effect.32 Furthermore, some viruses do Rotavirus
Vaccinia virus
not produce a cytopathic effect on infecting cells, or the Varicella-zoster virus
cytopathic effect that is produced is subtle and easily
missed. In these cases, the cultures will be reported as Single-Stranded DNA Virus Parvovirus
a false-negative result. Another disadvantage of using
Double-Stranded RNA Virus Rotavirus
viral cultures to detect and identify viral infections is
that the specimen must be collected in the acute phase Single-Stranded RNA Viruses Colorado tick fever virus
of the disease, that is, in the first 5 days of the illness, Coronavirus*
Coxsackie virus*
after which the amount of virus in body fluids decreases
Dengue virus*
significantly and may result in false-negative cultures. Ebola virus†
Nucleic acid amplification assays have become indis- Echovirus*
pensable in the clinical virology laboratory. Molecular Hepatitis A virus*
methods are well suited to targeting the various con- Hepatitis C virus
Hepatitis D virus
figurations of nucleic acids found in human pathogenic
Hepatitis E virus
viruses (Table 11.4). Target amplification assays such as Human T-cell leukemia virus‡
PCR, reverse transcriptase PCR (RT-PCR), quantitative Human immunodeficiency virus‡
PCR (qPCR), and transcription-mediated amplification Influenza virus
(TMA) as well as signal amplification assays such as Measles virus†
Mumps virus†
branched DNA (bDNA) amplification and hybrid capture
Norwalk virus, norovirus
are used in the clinical virology laboratory to diagnose Parainfluenza virus
or monitor viral infections. Table 11.5 summarizes the Poliovirus*
viruses for which nucleic acid amplification assays have Rabies virus
been developed along with the type of amplification pro- Respiratory syncytial virus
Rhinovirus*
cedure, the targeted genes, and clinical utility.
Rubella virus*
Molecular-based tests for HIV, Epstein–Barr virus Yellow fever virus*
(EBV), human papillomavirus (HPV), HCV, and BK/JC
viruses are frequently used in clinical laboratories. *Positive RNA; directly translated.
Human immunodeficiency virus (HIV) is an RNA †
‡
Negative RNA; complementary to the translated strand.
Retroviral replication requires a DNA intermediate.
virus that makes a DNA copy of genomic RNA using its
own virally encoded reverse transcriptase. There are two
types of HIV: HIV type 1, or HIV-1, and HIV type 2, or virus that are found in different locations in the world.
HIV-2. HIV-2 is a minor isolate found mainly in West There are four HIV subtypes, M (Major), O (Outlier),
Africa and is less pathogenic than HIV-1, causing more N (non-M and Non-O), and P. HIV group M causes 95%
latent infections. New strains continue to evolve. of the infections due to HIV around the world. Group
HIV rapidly mutates and recombines so that multi- M is further divided into eight clades (A, B, C, D, F,
ple groups then divide into “clades,” or subtypes, of the G, H, and J). Group M, clade B, is found most often in
TABLE 11.5 Nucleic Acid Amplification (NAA) Tests for Viruses
NAA Dynamic Range/

Virus Methodology Amplified Target Sensitivity Clinical Utility
Human PCR gag gene; 155 bp (HIV-1 400–750,000 copies/mL Viral quantitation
immunodeficiency RT-PCR NASBA group M [subtypes A-H], (standard) Disease prognosis
virus bDNA not HIV-2 or HIV-1 group O) 50–100,000 copies/mL Treatment monitoring
gag (similar to Amplicor) (ultrasensitive)
HIV-1 groups M, O, and N 176,000–3,470,000
pol; subtypes of group M copies/mL
(subtypes A–G, but not 75–500,000 copies/mL
group O)
Cytomegalovirus Hybrid capture Immediate-early antigen 1 1,400–600,000 Detect CMV DNA in organ
(CMV) PCR Major immediate-early copies/mL transplant and AIDS
NASBA qPCR antigen 400–50,000 copies/mL patients and congenitally
Major capsid Glycoproteins B and H infected infants
protein Detect HSV when Viral load determinations
asymptomatic or when
cultures are negative
Herpes simplex PCR Thymidine kinase Diagnosis of HSV

virus-1 and -2 qPCR DNA polymerase encephalitis and neonatal
(HSV) DNA-binding protein infections
Glycoproteins gb, gc, gd,
and gg
Epstein–Barr virus PCR EBNA1 100–1010 copies/mL EBV-associated

(EBV) qPCR LMP-1 malignancies
Detect EBV in asymptomatic
immunocompromised hosts
Human Hybrid capture L1 or E1 open reading frames 105 copies/mL Detection of HPV in
papillomavirus PCR endocervical swabs
(HPV) qPCR Differentiation of low-risk
and high-risk types
Monitoring women with
abnormal Pap smears
Hepatitis B virus PCR 1,000–40,000 copies/mL Prognosis and monitoring

bDNA 0.7–5,000 meq/mL of antiviral treatment
Hybrid capture 142,000–1,700,000,000 response
copies/mL (standard)
4,700–56,000,000
copies/mL (ultrasensitive)
Parvovirus B19 PCR Diagnosis of infections
Respiratory RT-PCR Fusion glycoprotein (F) gene Detection of RSV

syncytial virus Nucleoprotein (N) gene Differentiate between
(RSV) subgroups A and B
Continued on following page

TABLE 11.5 Nucleic Acid Amplification (NAA) Tests for Viruses (Continued)
NAA Dynamic Range/

Parainfluenza RT-PCR Hemagglutinin- Epidemiology

viruses neuraminidase conserved
regions
5’ noncoding region of F gene
Influenza viruses RT-PCR Conserved matrix (M) genes Diagnose infections

(influenza A and B) Characterize isolates
Nucleocapsid protein Can be type- and
(influenza A) subtype-specific
NS1 gene (influenza B)
Metapneumovirus RT-PCR Fusion (F) gene

RNA polymerase (L) gene
Coronavirus RT-PCR RNA polymerase gene Used to detect and

Nucleoprotein gene characterize the SARS virus
Norwalk virus RT-PCR RNA polymerase gene
Rotavirus RT-PCR VP7 gene

VP4 gene
Hepatitis C virus RT-PCR 5’ untranslated region (UTR) 600–800,000 IU/mL

bDNA 5’ UTR and core protein gene 3,200–40,000,000
copies/mL
West Nile virus RT-PCR Variety of gene targets based 0.1–1 PFU Used for diagnosis and
NASBA on genome of type strain surveillance
NY99
0.01 PFU
Rubella virus RT-PCR Surface glycoprotein, E1, gene Variable; sensitivity of Primarily for fetal diagnosis
3–10 copies is best Used for diagnosis when
serum is not available
May be used to confirm
positive serological results
Mumps virus RT-PCR Hemagglutinin, Differentiate strains

neuraminidase, P, SH, and F
genes
Measles virus RT-PCR M, H, F, N When culture is not

practical or genotyping is
required for diagnosis of
MIBE or SSPE
Differentiation of vaccine
and wild-type strains
TABLE 11.5 Nucleic Acid Amplification (NAA) Tests for Viruses (Continued)
NAA Dynamic Range/

Enteroviruses RT-PCR Conserved 5’ nontranslated Performed on CSF to rule

(group A and B region out enteroviral meningitis
Coxsackieviruses,
echoviruses, and
others)
BK virus PCR Large T protein Diagnosis of BK virus

(polyomavirus) nephritis
MIBE, Measles inclusion body encephalitis; PFU, plaque-forming units; SSPE, subacute sclerosing panencephalitis.
the United States and Europe. Group O HIV is found The amount of HIV or viral load is used as a marker
primarily in West Africa, and group N is found in Cam- for disease prognosis as well as to track the efficacy of
eroon. To infect host cells, the HIV surface molecules antiretroviral therapy. The goal of antiretroviral therapy
gp120 and gp41 interact with CD4, a molecule that is is a viral load below 50 copies/mL of blood, where it
expressed primarily on the surface of helper T lympho- is undetectable by most methods. Patients who maintain
cytes but is also found on macrophages, dendritic cells, viral levels at fewer than 10,000 copies/mL in the early
and other antigen-presenting cells. Chemokine receptors, stages of the infection are at decreased risk of progres-
in particular CCR5, on dendritic/Langerhans’ cells and sion to acute immunodeficiency syndrome (AIDS).33
macrophages/monocytes, and CXCR4 on CD4+ T cells Patients who are effectively treated with antiretrovi-
form a complex with CD4 on the cell surface and ral therapy will have a significant reduction in viral load
also engage gp120. After attachment to host cells via 1 week after the initiation of therapy. The lack of a sig-
CD4-gp120 binding, the virus enters the cell, where nificant decrease in viral load during this time indicates
reverse transcriptase makes cDNA from viral RNA. The the lack of efficacy. Highly active antiretroviral therapy
cDNA integrates into the host DNA, where it either per- (HAART), consisting of two reverse transcriptase
sists in a stage of latency as a provirus or is replicated inhibitors combined with a protease inhibitor or a non-
actively. Transcription and translation of viral peptides, nucleoside reverse transcriptase inhibitor, reduced viral
as well as production of viral RNA, are performed loads below the detection limits of even ultrasensitive
by cellular components under the direction of virally assays.34 In patients receiving HAART, 2 log10 decreases
encoded regulatory proteins (i.e., tat, rev, nef, and vpr). in viral load have been documented. Viral load testing is
HIV infection is identified as antibodies specific for performed in conjunction with determining CD4 counts.
HIV in an EIA and by confirming the specificity of In general, but not always, viral load and CD4 counts
detected antibodies for HIV products in a western blot or are inversely proportional; that is, the higher the viral
a qualitative RNA probe assay. The antigens used in the load, the lower the CD4 count.
western blot tests may differ depending on the country HIV in patient samples can also be detected by
of origin. For infants who have maternal IgG and for PCR of integrated DNA, nucleic acid sequence–based
patients suspected of incubating HIV in whom antibody amplification (NASBA), and bDNA (see Table 11.5).
tests are negative, antigen detection tests measure the Table 11.6 compares the advantages and disadvan-
amount of HIV p24 antigen. Quantitative nucleic acid tages of each of these assays for determining HIV viral
amplification assays are performed after an HIV diag- load. Viral load should be determined before therapy is
nosis to determine how actively the virus is replicating started, 2 to 8 weeks after therapy initiation to see the
(viral load), when to start antiretroviral therapy, and initial response, and then every 3 to 4 months to assess
when to monitor the efficacy of treatment. therapeutic effectiveness.
TABLE 11.6 Advantages and Disadvantages TABLE 11.7 Test Performance Features
of the Nucleic Acid Amplification Methods for Viral Load Measurement
for HIV Viral Loads
Characteristic Description
Test Advantages Disadvantages
Sensitivity Lowest level detected at least 95% of
bDNA High throughput No internal control the time
Broad dynamic range False-positive
Applicable for group results reported Accuracy Closeness of measured value to a
M subtypes A–G standard or known value
Amplicor Internal control Limited dynamic Precision Reproducibility of independently

RT-PCR Good specificity range determined test results
NASBA Broad dynamic range Does not detect all Specificity Negative samples are always negative,
Performed on many non-B subtypes and positive results are true positives
specimen types and
Linearity A serial dilution of standard curve
volumes
closely approximates a straight line
Flexibility Accuracy of measurement of virus

Results among methods are increasingly comparable, regardless of sequence variations
such as viral loads determined by bDNA and RT-qPCR.35
Even so, it is still recommended that the same method to
determine viral loads is used when monitoring patients for viral load testing. It is established by the calibra-
over time. Results are expressed consistently as inte- tion of assays to a common standard. Quantitative tests
gers (copies/mL or IU/mL), log-transformed results (log must also have accuracy that is a true measure of the
IU/mL), scientific notation (a × 10b IU/mL), or a com- viral level over a range of values. The viral load levels
bination of these. CDC guidelines recommend report- established by the DHHS and the International AIDS
ing in integers and log-transformed copies. Quantitative Society for the initiation of therapy must be consistently
HIV-1 RNA testing in plasma has been the standard for identified in independent laboratories as accurately as
monitoring drug therapy and HIV disease progression. possible. Quantitative PCR methods offer linear mea-
The Multicenter AIDS Cohort Study, an ongoing project surement over a wider range than other methods, which
monitoring the clinical histories of treated and untreated precludes the requirement for dilution of high-titer spec-
HIV-infected men, has described laboratory mea- imens before analysis. The precision or reproducibility
surements of viral load and clinical disease. The U.S. of the test used is also important for establishing sta-
Department of Health and Human Services (DHHS) rec- tistically significant differences in the viral load over
ommends an objective of maximal suppression of viral a serial testing period. The DHHS defines a minimally
replication down to undetectable levels by sensitive significant change as a threefold increase or decrease in
analysis. viral load/mL plasma. And the high specificity of a test
The first commercially produced tests could detect gives confidence that a positive result is truly positive.
viral loads down to 400 to 500 copies/mL plasma; All subtypes of virus should be detected with equal effi-
however, suppression to fewer than 20 copies/mL plasma ciency to avoid under- or overestimating viral loads of
was associated with longer response to therapy than certain subgroups.
suppression below 500 copies/mL, which emphasizes HIV RNA in the same patient will not change much
the importance of a highly sensitive assay with a very over time (approximately 0.3 log10 unit) as long as the
low limit of detection for optimal treatment strategy and patient is clinically stable and antiretroviral therapy has
patient care. In addition to sensitivity, all test methods, not begun or changed. In order to be clinically relevant,
including HIV tests, should have certain characteris- viral load changes from one determination to another
tics (Table 11.7). Accuracy is an important requirement must vary by threefold (0.5 log10 unit). HIV-positive
patients may experience transient increases in viral loads Mutations found by genotyping are generally divided
when they have other infections or receive vaccinations, into two groups: primary resistance mutations and
but levels will return to baseline within a month. Profi- secondary resistance mutations. Primary resistance
ciency testing is available from the College of American mutations are those that are specific for a particular
Pathologists (CAP; Northfield, IL) and the CDC. The drug, reduce the susceptibility of the virus to that drug,
World Health Organization (WHO) has an HIV-1 RNA and appear in the viral genome shortly after treatment
reference standard. with that agent has begun. The mutated enzyme is gen-
Error-prone replication of HIV by its reverse tran- erally not as active as the normal enzyme, so viral repli-
scriptase and recombination between co-infecting strains cation is decreased, but it still occurs. As treatment with
generates new HIV sequences, which can affect the anti- the drug continues, secondary or compensatory muta-
viral drugs, including protease inhibitors, nucleoside tions occur that try to recover the ability of the virus to
analogs, reverse transcriptase inhibitors, and inhibitors replicate at a normal rate. The secondary mutations do
of viral integration. Genotyping is used to monitor the not affect the susceptibility of the virus to the drug, but
development of this antiretroviral drug resistance. Most rather help the virus replicate in the presence of the drug
of the antiretroviral drugs target the reverse transcriptase when one of its replication enzymes is not 100% func-
and protease enzymes, so these are the genes that are tional. Once a resistance genotype has been identified,
most often examined in genotyping procedures. the drug therapy of the patient should be changed as
To perform genotyping, viral RNA is extracted, and soon as possible to avoid the development of secondary
PCR is used to amplify the whole protease gene and mutations in the virus.
part of the reverse transcriptase gene. The products are The results of genotyping procedures are reported by
analyzed for the presence of mutations by sequencing, listing the mutations that have been identified in the pro-
hybridization onto high-density microarrays, or reverse tease and reverse transcriptase genes and the impact those
hybridization. Sequencing is performed most often, and mutations will have on each drug: no evidence of resis-
there are commercially available kits for Sanger sequenc- tance, possible resistance, resistance, or insufficient evi-
ing. Using these genotyping methods for resistance dence. The mutations are indicated by reporting a change
monitoring, however, is a high-cost and low-throughput in the amino acid that is coded by the changed codon,
method. Next-generation sequencing (NGS) along with where the wild-type amino acid is written, followed by
simpler sample collection and storage matrices (e.g., the position of the codon that is changed, followed by the
dried blood spots36) will facilitate and broaden resistance new amino acid. For example, a mutation in codon 184 of
monitoring as well.37 the reverse transcriptase gene from ATG to GTG results
To evaluate resistance, viral sequences are com- in an amino acid change from methionine to valine, or
pared with a reference sequence to identify the muta- M184V. This particular mutation makes the virus resis-
tions present. After the mutations have been identified, tant to the cytidine analog, lamivudine. Another muta-
their significance in terms of the impact on antiretroviral tion, Q151M, located at the dNTP-binding site of reverse
therapy is assessed, and this is generally accomplished transcriptase, confers resistance to reverse transcriptase
through computer algorithms. Resistance mutations inhibitors while maintaining sensitivity to lamivudine.
have been well characterized for individual agents, but As with all other molecular-based assays, HIV geno-
HIV-positive patients are more often on cocktails of typing procedures should employ adequate quality and
drugs rather than one drug. Therefore, the impact of a contamination controls. The sensitivity of the methods
mutation on multiple drugs is also considered. In addi- for detecting a minority of virions that contain muta-
tion, if a single virus has multiple mutations, the inter- tions in the midst of a majority of wild-type virions is
pretation of more than one mutation in the context of an important consideration. The sensitivity of the auto-
the others and with respect to multiple drugs becomes mated sequencing methods has been reported to be 20%,
more complex. Computer algorithms are used to analyze and NGS has a reported sensitivity of less than 1%.39
genotypes, taking into account primary and secondary Proficiency testing is provided by the CAP, and indepen-
mutations, cross-resistance, and the interactions between dent control materials for use in genotyping assays are
mutations that can affect resistance.38 commercially available.
Herpes viruses are frequent sources of human infec- infection is characterized by the presence of EBV-en-
tion. At least 25 viruses comprise the family Herpes- coded RNA (EBER). The virus can reactivate and is
viridae. Several herpes virus types frequently infect commonly found in the saliva of infected persons. EBV
humans, including herpes simplex virus (HSV), CMV, infection is associated with a variety of benign and
EBV, and varicella-zoster virus (VZV). In addition to malignant lesions, including Hodgkin disease, non-Hod-
causing overt disease, herpes viruses may remain silent gkin lymphoma, Burkitt lymphoma, gastric carcinoma,
and be reactivated many years after infection. and nasopharyngeal carcinoma. Although EBV is
HSV has a relatively large viral genome encoding present in these conditions, it is likely not the sole cause
at least 80 protein products. About half of these proof disease.
teins are involved in the interaction with the host cell Laboratory testing for EBV infection has been per-
or immune system. The other half control viral struc- formed by immunohistochemistry for EBV proteins
ture and replication. There are two types of HSV, HSV in tissue and testing serum for antibodies to EBV-
type 1 (HSV-1) and HSV type 2 (HSV-2). HSV-1 mainly associated antigens, including the viral capsid antigen,
causes cold sores or fever blisters; HSV-2 mainly causes the early antigen, and the EBV nuclear antigen (EBNA).
genital sores but can also cause mouth sores. HSV tests Confirmation may be done by differentiation of IgG and
are usually done for genital sores, although body fluids, IgM subclasses to the viral capsid antigen. EBER tran-
including blood, urine, tears, amniotic fluid, lavages, and scripts are primary targets for tissue analysis. Repeated
spinal fluid, may also be tested. or unique sequences in EBV DNA are targets of in situ
HSV was detected by viral culture, antigen, and anti- hybridization; however, the sensitivity of DNA probes
body tests before the application of PCR. Viral cultures is lower than those for EBER, due to the abundant pres-
sometimes had to be performed more than once because ence of the EBER transcripts.
the virus might not be detected at all times of infection. Southern blot analysis of EBV DNA, first described
The HSV antigen test was performed on a slide smear of in 1986,40 was based on variable numbers of terminal
material from the sore. Viral antigens are detected with repeat sequences at the ends of each EBV DNA mole-
labeled antibodies. The antibody test was performed on cule. Each infecting genome can contain up to 20 ter-
blood to detect antibodies to the viral antigens. Anti- minal repeat sequences. When EBV DNA is cut with
bodies may not be detectable 2 weeks to 3 months after BamH1, the resulting fragments vary in size, depending
initial infection; however, once the infection occurs, anti- on the number of repeat sequences. The fragments were
bodies remain in the person for life. Western blot tests visualized with a probe complementary to the variable
could distinguish HSV-1 and HSV-2 using type-specific repeat.
antigens to detect the corresponding type-specific anti- Amplification methods are now used for detect-
bodies. Type-specific testing is important for prognosis ing EBV in blood, body fluid, or tissue samples. EBV
and patient counseling. DNA can be amplified using primers complementary
PCR tests also offer the advantage of distinguishing to conserved EBV sequences, and strain typing can be
HSV-1 from HSV-2 directly without culturing. Methods achieved by amplification of polymorphic regions of
include standard PCR and quantitative PCR target- the viral genome. Quantitative PCR is used to deter-
ing type-specific HSV genes (Table 11.5). One of the mine EBV viral load in blood using a reference inter-
most common human viruses, EBV is a member of the nal control. A reported quantitative range of analysis is
herpes virus family. Most people become infected with 750 to 106 IU/mL.41 As with other such tests, the virus
EBV sometime during their lives. Children can become may still be present below the level of detection.
infected with EBV once they lose maternal antibody Another member of the herpes virus family is CMV.
protection, and most adults in the United States between Most people have been infected with CMV without
ages 35 and 40 have likely been infected with EBV. The obvious illness. Like other herpes viruses, CMV can
first infection during adolescence or young adulthood go dormant and reactivate. If symptoms occur, they are
causes infectious mononucleosis. mild, except in immunocompromised individuals. The
EBV establishes a permanent dormant infection in virus is shed in blood, urine, saliva, and other body
cells of the throat, blood, and immune system. Latent fluids but dies quickly outside of the host.
Molecular detection of CMV is performed on cell- stage. The development of chronic infections is due to
free plasma or other fluids. DNA is extracted and the antigenic envelope proteins that elicit the produc-
amplified by PCR using primers targeting the CMV tion of antibodies. These proteins are encoded by hyper-
polymerase (UL54) or glycoprotein B (gB) gene in variable regions much like antibody genes themselves,
regions that do not share sequence homology with other resulting in extensive variation in the envelope proteins
herpes viruses. Internal controls are included to avoid and the escape of the virus from antibodies.
false-negative interpretation due to PCR inhibition. The The initial approaches to HCV analysis are similar to
level of detection of standard laboratory assays is such those for HIV. Serology is used to detect the presence
that no PCR product will be produced from normal of antibodies against HCV. If the patient has HCV anti-
plasma, even from people previously exposed to CMV. bodies, the specificity of the antibodies for HCV anti-
Automated amplification and detection of CMV has an gens is measured by western blot, where the presence
analytical sensitivity as low as one viral copy per micro- of antibodies with multiple HCV specificities confirms
liter. Quantitative PCR can detect down to 40 genome the diagnosis.
copies of virus per microliter of blood. PCR assays A variety of nucleic acid amplification assays for
may be affected by the presence of mutations in the the qualitative detection and quantitation of HCV are
target genes that interfere with primer hybridization. available, including RT-PCR, transcription-mediated
These mutations may also confer resistance to antiviral amplification, and branched DNA. The qualitative HCV
therapy. RNA assays are performed on patients with positive
VZV (HHV3) is a herpes virus that infects younger HCV antibody results to confirm active infection or
and older humans. VZV is the causative agent of chick- on immunocompromised patients (who are often co-
enpox and shingles (also called herpes zoster). The virus infected with HIV) in whom HCV infection is suspected
has a large genome containing over 70 open reading but antibody tests are negative. The quantitative HCV
frames (ORFs). Polymorphisms in the ORF are used to RNA assays are used as for HIV to determine the viral
determine the strain variations and genotype of viruses load and to monitor viral replication in response to anti-
from different geographical areas. An attenuated live viral therapy. The viral load and the HCV genotype are
virus, VZV Oka, developed by selection in cell cul- used to determine the therapeutic protocol, both type of
tures, is the parental strain source of a VZV vaccine, drug(s) as well as duration.
which contains a mixture of genotypically distinct viral Six genotypes (1a/1b, 2, 3, 4, 5, and 6) and more than
variants. 50 subtypes of HCV have been identified. The geno-
VZV is neurotropic, remaining latent in nerve cells type of HCV present in a given patient determines the
and possibly reactivating years after primary infections treatment protocol used on that patient, as particular
to result in shingles, or palsy. It is found in more than genotypes are associated with certain antiviral resistance
90% of young people living in temperate climates. There patterns. The HCV genotype is determined by analyzing
is no animal reservoir for VZV. Clinical tests include the core and/or 5′ untranslated regions of the genome.
enzyme-linked immunosorbent assays and PCR methods Laboratory methods available for HCV genotyping are
for detection of antibodies to VZV in serum. Qualitative PCR with restriction fragment length polymorphism
PCR methods to detect VZV include PCR with hybrid- (RFLP) analysis and reverse hybridization and direct
ization or direct gel electrophoresis. Quantitative PCR DNA sequencing. A PCR with melt-curve method has
kits include internal controls to detect amplification also been described.42
inhibition. It has been observed that patients who have a 2 log10
HCV is an enveloped, single-stranded RNA virus of decrease in HCV RNA 12 weeks after treatment begins
the Flaviviridae viral family. HCV causes viral hepatitis have a 65% chance of responding, defined by the lack
and cirrhosis and is also associated with causing hepato- of detection of HCV RNA in qualitative assays where
cellular carcinoma. The virus is transmitted parenterally the detection limit is 50 to 100 copies of virus/mL of
like HIV. Acute infections are often asymptomatic and plasma. Patients who do not have a 2 log10 decrease in
are rarely associated with jaundice; thus, patients with HCV RNA 12 weeks after treatment begins have less
acute HCV infections are usually not detected at this chance of responding. Determining the genotype of the
virus is also critical to predicting treatment outcomes; percentage of HPV DNA infected cells that are pre-
for example, genotypes 2 and 3 will respond better to cancerous. The PCR genotyping detects 37 high- and
particular treatments than genotype 1. low-risk HPV types by hybridization of PCR products
HPV is a double-stranded DNA virus recognized as to immobilized probes for each of the mutations. Direct
oncogenic in several human cancers. There are over sequencing may also be used to detect more HPV types.
200 HPV types based on sequences of the viral genome Methods may differ, not only in the sensitivity to
compared with known HPV genomes. Five evolution- target viruses but with the ability to detect concurrent
ary HPV genotype groups (α, β, γ, μ, and ν) have been infection with different HPV types. In all cases, patient
defined. The largest, the α group, contains 64 types that management decisions reflect patients’ overall cytology
mainly infect mucosal epithelia in the anogenital tract history and other risk factors in addition to the presence
and include the high risk (HR) types (HPVs 16, 18, or absence of high-risk HPV types.
31, 33, 35, 39, 45, 51, 56, 58, 59, 68, 73, 82) that have Respiratory viral infections are a major cause of hos-
been classified as oncogenic and are found to cause ano- pitalizations in young children and elderly people in
genital cancers. The next largest group is the β-group the United States. Over a dozen respiratory pathogens
HPVs that contains more than 50 characterized types (viral and bacterial) are commonly encountered in the
that mainly infect cutaneous epithelia. The β group and medical laboratory. Treatment and control of the spread
ultraviolet (UV) radiation exposure are associated with of infection will depend on which of these are infecting
nonmelanoma squamous cell carcinomas, a common a patient. For viral infections, molecular methods have
human cancer. HPVs of the remaining three groups (γ, been designed to detect multiple species in single anal-
μ, and ν) normally cause only benign disease.43 HPV yses. For example, a real-time PCR method can detect
HR types are responsible for over 95% of cervical squa- influenza A, influenza B, and RSV. Influenza A subtyp-
mous carcinoma. The presence of high-risk HPV DNA ing by melt-curve analysis has been used as a screen-
in conjunction with an equivocal or ambiguous cytology ing method to detect the 2009 influenza A (H1N1) virus
result (atypical squamous cells of unknown significance from nasal swabs.
[ASC-US]) indicates an increased risk for having an
underlying cervical neoplasm. Persistent infection with
high-risk HPV may be the main risk factor for the devel- Advanced Concepts
opment of high-grade cervical neoplasia and cancer.
Women with normal cervical cytology who are negative Amplification methods that target the HPV
for the high-risk HPV types are at low risk for having or L1 gene may not detect virus that has integrated
developing cervical precancerous lesions. into the host DNA because that gene region may
It is difficult to culture HPV in the laboratory, so be lost, causing false-negative results. Coin-
detection relies on molecular testing. Two approaches to fection with two or more HPV types will lower
molecular detection of HPV are hybridization methods overall PCR efficiency for each type or selectively
and amplification methods. The latter methods include amplify only one type, also resulting in false-
target amplification, such as PCR, and signal amplifi- negative results.
cation, such as hybrid capture. Hybridization tests can
detect a minimum of 4,000 to 5,000 viral genomes.
These tests also have the capacity for subtyping. HPV RSV is detected using several assays. An ASR for RSV
expresses its early genes, E1 through E8, shortly after A + B RNA uses NASBA technology. A bead array
host infection. The E6 and E7 genes are overexpressed technology can simultaneously detect RSV A and B,
after integration of oncogenic genotypes of the HPV influenza A, nonspecific influenza A, H1, H3, influ-
genome in the host genome. E6 and E7 gene products enza B, parainfluenza 1, parainfluenza 2, parainfluenza
are involved in the cellular transformation to cervical 3, metapneumovirus, rhinovirus, and adenovirus. The
cancer. Measurement of E6 and E7 mRNA expression test includes an MS-2 bacteriophage internal control
in specific cell types identified by flow cytometry is a and a lambda bacteriophage positive control. It aided
specific indication of cellular transformation in the small in the detection of 2009 influenza A/HIN1 (swine flu)
but could not identify the hemagglutinin gene of the

Mycology
2009 influenza A/H1N1 directly.44 Seasonal and H1N1
flu viruses develop resistance to antiviral agents through Fungi are among the most ubiquitous microorganisms
sequence alterations.45 These mutations can be detected causing clinical infection. Traditional methods of iden-
by direct sequencing. Digital PCR methods have also tification by phenotype have become more difficult
been developed to detect the frequently occurring alter- with the expanding diversity of organisms. Molecular
ation H275Y in the neuraminidase gene (H274Y in methods, particularly sequencing and PCR, have allowed
N2 nomenclature).46 for the detection and typing of fungi with greater sensi-
BK and JC viruses are human polyomaviruses, the tivity, specificity, and speed.
primate counterpart of which is SV40. BK, JC, and Fungi are important causes of human disease, espe-
SV40 share sequence and antigenic homology. They are cially in immunocompromised patients. In addition,
double-stranded DNA viruses with 5,000-bp genomes laboratory-acquired infections from fungi are a major
encoding 5 transcripts. Most polyomavirus infections are risk for laboratory personnel. Fungal infections are most
subclinical in adults. Infected children develop respira- often diagnosed by direct staining methods and isolation
tory symptoms, and some have cystitis. Several reagent of the causative agent in culture. As for other organisms,
sets are available for detection of BK or JC viruses in the traditional smears and cultures are affected by sensitiv-
clinical laboratory, including quantitative PCR tests.47 ity, organism viability, and the length of time required
for the organism to grow. Despite these problems, direct
smears and isolation of fungi are still the major methods
Mass Spectrometry
for detecting fungi in clinical samples.
Application of mass spectrometry to bacteriology has Fungi growing in culture are typically identified by
been extended to virology to a limited degree. Viruses their microscopic and macroscopic morphologies or
have low concentrations of high-molecular-weight pro- by using fluorescent antibodies. For some fungi, such
teins that are more difficult to assess by peptide pro- as Histoplasma, Blastomyces, Coccidioides, and Cryp-
files. Confounding viral detection is cell debris from the tococcus neoformans, gene probes have been devel-
cultures in which the viruses were cultivated. MALDI oped to confirm the identity of the organism growing
analysis of PCR-amplified viral nucleic acid has been in culture.51 Using DNA probes for these organisms is
successfully applied to clinical detection of HSV, HCV, faster and less hazardous than determining the micro-
HPV, enteroviruses, and respiratory viruses.48 Multiplex scopic morphology.
PCR with MALDI (PCR-mass assay) has been shown to Broad-range PCR and subsequent analysis are also
identify multiple viruses or multiple viral subtypes in a used for clinical analysis of fungi. In these approaches,
single test.49 primers anneal to DNA sequences that are common to
PCR-based genotyping of frequently rearranged most of the clinically relevant fungi, such as Candida,
viruses such as influenza where recombinant viruses Aspergillus, Rhizopus and other Zygomycetes, and
arise from coinfection with distinct viral stains has Histoplasma and other dimorphic fungi. Once the
proven more difficult. PCR amplification in these cases sequences are amplified, hybridization to species-specific
becomes problematic as primer-binding sights are lost probes or sequencing is used to identify the fungus to the
or changed in the recombinant strains. These reassorted genus or genus and species level. In-house-developed
flu virus strains are also difficult to detect by immuno- real-time PCR methods are also used for direct iden-
logical methods as the antigenic determinants become tification of Aspergillus and Pneumocystis carinii.
altered. Characterization of the type of virus is impor- Blastomycetes, Coccidioides immitis, and Histoplasma
tant for early intervention and prevention of infectious capsulatum are detected with direct probe hybridiza-
spread. For this application, mass spectrometry has been tion assays. The probe methods can also be multiplexed.
shown to successfully genotype viruses using whole- PFGE is used in laboratory-developed methods for
virus protein digests.50 Human influenza and parainflu- molecular typing of yeasts.
enza viruses can be subtyped and lineages traced by this Molds can be typed by PCR and sequencing of ITS
method. regions or 28S rRNA. ITS sequences of DNA extracted
from 24 to 48 mold cultures are compared with those of Recognition of these issues has made the develop-
reference strains. This method is used in industrial and ment of molecular-based assays for parasite detection
environmental settings to positively identify Paecilo- and identification more practical. Nucleic acid isolation
myces species, which are saprophytic filamentous fungi for molecular testing has been addressed by using several
found in soil and as air and water contaminants. Paeci- methods, including extraction and extraction-free,
lomyces lilacinus and Paecilomyces variotii are increas- filter-based protocols for preparation of DNA templates
ing causes of opportunistic human infections generally from oocysts and microsporidia spores. PCR methods
associated with the use of immunosuppression therapy, are modified to accommodate sequence content, for
saline breast implants, or ocular surgery. Although these example, low GC content in malaria.
two species present differences in their in vitro suscepti- PCR assays have been developed for the detection of
bility to antifungal agents, requiring identification before Trypanosomes, Plasmodia, Toxoplasma, Entamoebae,
treatment, molecular testing in the clinical laboratory is and Cryptosporidium in hosts and water sources. Multi-
not well developed. plex qPCR methods can also measure infection rates and
MALDI-TOF application to detection of fungi has organism loads in hosts and nonhuman vectors (carriers
been limited by their biological complexity and different of infection).54
fungal growth phases. Furthermore, their thick cell walls In-house-developed real-time PCR methods are used
require chemical and physical disruption methods. Cell in clinical laboratories for identification of Babesia,
walls are extracted with trifluoroacetic acid, or formic Trichomonas microti in blood and from ticks, Enceph-
acid and acetonitrile, possibly followed by physical dis- alitozoon species microsporidia, and Trichomonas vag-
ruption with beads. inalis. Multiplex PCR methods have been designed to
Mass spectrometry methods for identification of S. simultaneously detect multiple parasites, for example,
cerevisiae were first developed in 2001.52 Reproducible multiplex real-time PCR assays for detection of Entam-
spectra have since been observed for other yeasts includ- oeba histolytica, Giardia lamblia, and Cryptosporid-
ing Candida and Cryptococcus. MALDI-TOF may also ium parvum simultaneously or E. histolytica, Giardia
offer a reliable, rapid approach to testing for fungal intestinalis, and Cryptosporidium spp. simultaneously
sepsis.53 in stool samples. The multiplex PCR methods also
include an internal control to determine the efficiency
of the PCR and detect inhibition in the sample, which
Parasites
is likely in stool samples. The development of mul-
Parasites are typically detected and identified by mor- tiplex PCR assays to detect multiple parasites in stool
phology directly in clinical specimens, a method of samples is extremely useful. First, multiple parasites
diagnosis subject to false-negative results in cases of can cause diarrhea, and morphology is the only way to
low organism concentrations and depending greatly on differentiate between causative agents. Second, patients
appropriately trained personnel. Molecular-based testing can have multiple intestinal parasites at the same time,
for parasites has been limited mainly because parasites and laboratory detection of the presence of all parasites
are not a major cause of disease in developed coun- is important. Finally, multiple parasites are transmitted
tries. Increasingly, however, travelers from parasite- through the same source; thus, detection of all parasites
endemic countries bring parasites to developed countries and appropriate control measures will reduce large-scale
and serve as a reservoir for transmission. In the labo- outbreaks.
ratory, nucleic acid template preparation from oocysts
and spores of protozoan parasites is complicated by the
nature of the organism and its resistance to disruption ANTIMICROBIAL AGENTS
and lysis. Parasites found in complex matrices such as
stool samples present a further difficulty due to inhi- Antimicrobial agents are of two types, those that
bition of PCR and other enzymatic assays. However, inhibit microbial growth (-static, e.g., bacteriostatic,
expertise in identifying parasites by morphology is fungistatic) and those that kill organisms outright
declining, demanding alternative methods of surveil- (-cidal, e.g., bacteriocidal, fungicidal). Antimicrobial
lance and detection of these organisms. agents for use in clinical applications are designed to be
Essential
TABLE 11.8 Mode of Action metabolism
of Antimicrobial Agents Cell wall integrity
Protein
synthesis
Mode of Action Examples
Disrupts cell wall synthesis Beta-lactams (penicillins and

or integrity cephalosporins)
Glycopeptides (vancomycin)
Disrupts cell membrane Polymyxins (polymyxin B)

structure or function Bacitracin Membrane
integrity
Inhibits protein synthesis Aminoglycosides (gentamicin)
Tetracyclines
Macrolides (erythromycin)
Lincosamides (clindamycin)
Nucleic acid
Inhibits nucleic acid Quinolones (ciprofloxacin) metabolism
synthesis or integrity Metronidazole FIGURE 11.5 Sites of antimicrobial action. Depending on
Inhibits metabolite Sulfamethoxazole the type of organism, several structures can be affected by anti-
synthesis Trimethoprim microbial agents. All of these are essential for cell growth and
survival.
selective for the target organism with minimal effect on antibiotics such as vancomycin can lead to the devel-
mammalian cells. These agents are also intended to dis- opment of resistant clones of organisms. These clones
tribute well in the host and remain active for as long as may persist in low numbers below the detection levels
possible (long half-life). Ideally, the agents should have of routine laboratory sensitivity testing methods.
-cidal (rather than -static) activity against a broad spec-
trum of microorganisms.
Another way to classify antimicrobial agents is by Advanced Concepts
their mode of action (Table 11.8). The ultimate effect of
these agents is to inhibit essential functions in the target The first antibiotics isolated were natural secretions
organism (Fig. 11.5). A third way to group antimicrobial from fungi and other organisms. Synthetic mod-
agents is by their chemical structure. For example, there ifications of these natural agents were designed
are two major types of agents that inhibit cell wall syn- to increase the spectrum of activity (ability to
thesis, the β-lactams with substituted ring structures and kill more organisms) and to overcome resistance.
the glycopeptides. For example, cephalosporins include such first-
generation agents as cephalothin and cefazolin that
are active against Staphylococcus, Streptococcus,
Resistance to Antimicrobial Agents
and some Enterobacteriaceae. A second generation
Microorganisms naturally develop defenses to antimi- of cephalosporins—cefamandole, cefoxitin, and
crobial agents. At the molecular level, resistance can cefuroxime—is active against more Enterobacteria-
arise from alerted target binding, active extrusion, or ceae and organisms resistant to β-lactam antibiotics.
inaccessibility of the drug or microbial enzymes that A third generation—cefotaxime, ceftriaxone, and
inactivate the drug. Multidrug-resistant organisms may ceftazidime—is active against P. aeruginosa as
have one or more of these characteristics. A single- well as many Enterobacteriaceae and organisms
nucleotide variant in a drug target or transport protein resistant to β-lactam antibiotics. The fourth gener-
can result in resistance. Resistant Staphylococcus, Pseu- ation, cefepime, is active against an extended spec-
domonas, and Klebsiella spp. are becoming common- trum of organisms resistant to β-lactam antibiotics.
place in health-care institutions. Long-term therapy with
TABLE 11.9 Resistance Mechanisms TABLE 11.10 Genes Conferring Resistance

to Antimicrobial Agents in Particular Organisms
Examples of
Mechanism Example Agents Affected Antimicrobial Gene(s) Conferring
Organism Agent Resistance
Destruction/ β-lactamases β-lactams
modification of agent aminoglycosides Staphylococcus Oxacillin mecA
aureus
Elimination of agent Multidrug β-lactams,
efflux systems fluoroquinolones, Streptococcus Penicillin pbp1a and pbp1b
macrolides, pneumoniae
chloramphenicol,
trimethoprim Gram- β-lactams tem, shv, oxa, ctx-m
negatives
Altered cell wall Thick cell Vancomycin
structure walls that β-lactams Enterococcus Vancomycin vanA, vanB, vanC,
exclude agent vanD, vanE, vanG
Altered agent
binding sites Salmonella Quinolones gyrA, gyrB, parC, parE
Alternate metabolic Altered Sulfonamides, Mycobacterium Isoniazid katG, inhA

pathways enzymes trimethoprim tuberculosis
Rifampin rpoB
There are several ways in which microorganisms

develop resistance (Table 11.9). First, bacteria can
produce enzymes that inactivate the agent. Examples will eventually take the place of those without the muta-
of this resistance mechanism are seen in S. aureus and tion, which are less able to survive and procreate. This
N. gonorrhoeae that produce β-lactamase, an enzyme process is stimulated by antibiotic exposure, especially
that cleaves the β-lactam ring of the β-lactam antimicro- if the levels of antibiotics are less than optimal. For
bials such as the penicillins. Cleavage of the β-lactam example, S. aureus developed resistance to antibiot-
ring destroys the activity of penicillin, rendering the ics that target its penicillin-binding protein (PBP1) by
organism resistant to its antimicrobial action. Second, replacing PBP1 with PBP2a encoded by the mecA gene.
organisms produce altered targets for the antimicrobial PBP2a found in methicillin-resistant S. aureus (MRSA)
agent. Mutations in the gene encoding a penicillin- has a low binding affinity for methicillin.
binding protein—for example, a change in the struc- Another genetic resistance mechanism is the acqui-
ture of the protein—render penicillin unable to bind to sition of genetic factors from other resistant organisms
its target. Finally, bacteria exhibit changes in the trans- through transformation with plasmids carrying resis-
port of the antimicrobial agent either into or out of the tance genes or transduction with viruses carrying resis-
cell. An example of this mechanism is seen in gram- tance genes. Genetic factors can also be transferred
negative bacteria that change their outer membrane from one bacterium to another by conjugation. Genet-
proteins (porins) in order to decrease the influx of the ically directed resistance can pass between organisms
antimicrobial agent. If the agent cannot get into the cell of different species. For example, MRSA (vancomycin-
and bind to its target, then it is not effective in inhibiting sensitive S. aureus) can gain vancomycin resistance
or killing the bacterium. from vancomycin-resistant Enterococcus faecalis. Van-
All these resistance mechanisms involve a genetic comycin and other glycopeptides act by preventing the
change in the microorganism (Table 11.10). These cross-linking of the peptidoglycan, thereby inhibiting
genetic changes are most commonly brought about by cell wall production. Several genes have been found in
mutation and selection processes. If a mutation results in enterococci that encode altered binding proteins: vanA,
a survival or growth advantage, cells with the mutation vanB, vanC, vanD, vanE, and vanG. The expression of
ORF 1 ORF 2 vanA vanS vanH vanA vanX vanY vanZ
Tn1546
58K VRSA
plasmid
FIGURE 11.6 Vancomycin-resistant S. aureus (VRSA) plasmid carrying transposon Tn1546 with vancomycin resistance genes.
vanA and vanB is inducible and transferred from cell to resistance for a given organism and antimicrobial agent
cell by plasmids carrying vancomycin resistance genes pair. The determination of MICs to detect antimicro-
on a transposon55 (Fig. 11.6). The resulting vancomycin- bial resistance is a phenotypic method. Although MIC
resistant S. aureus (VRSA) uses lactic acid instead of methods are well established, and the results are gener-
alanine to build its cell wall. The VRSA cell wall, then, ally reliable with regard to in vivo effectiveness of an
does not contain the target structure (D-ala-D-ala) for agent for an organism, the results are sometimes diffi-
vancomycin. Development of resistance to particular cult to interpret and the procedures are time-consuming,
drugs occurs with particular mutations: rpoB mutation taking at least 48 hours after the specimen is collected.
is associated with rifampin resistance, and mutations in Molecular methods that detect genes directly involved
katG, inhA, ahpC, and ndh genes are associated with in the resistance of an organism to a particular agent offer
resistance to isoniazid.56 a more straightforward prediction of antibiotic resis-
tance. There are four reasons for using molecular-based
Molecular Detection of Resistance methodologies. First, when an organism has an MIC at
or near the breakpoint of resistance, detection of mutated
Susceptibility to antimicrobial agents is determined by
genes contributing to resistance would be irrefutable
phenotypic or genotypic methods. The phenotypic devel-
evidence of the potential ineffectiveness of the agent.
opment of resistance is detected by performing in vitro
Second, genes involved in the resistance of organisms to
susceptibility testing. Testing for altered sensitivity to
antimicrobial agents can be detected directly in the clin-
antimicrobial agents is of clinical significance especially
ical specimen closer to the time of collection and save
when organisms persist in patients being treated with
the time required to isolate the organism and perform
antimicrobial agents that are generally considered effec-
phenotypic MIC determinations on isolated colonies.
tive against the particular isolate or when large numbers
With no requirement for culturing potentially dangerous
of organisms are observed in normally sterile fluids,
microorganisms, there is less chance of hazardous expo-
such as blood, cerebrospinal fluid, or urine. Phenotypic
sure for the technologist as well. Third, monitoring the
methods are generally used for aerobic bacteria, some
spread of a resistance gene in multiple isolates of the
mycobacteria, and yeast. For other organisms, such as
same organism is more useful in epidemiological inves-
viruses and filamentous fungi, phenotypic methods
tigations than following the trend in the MIC. Finally,
are not well standardized. Phenotypic methodologies
molecular methods are considered the gold standard for
include disk diffusion, broth dilution, and direct detec-
validation of new phenotypic assays.
tion of resistance factors such as β-lactamase.
Susceptibility testing measures the minimum inhib-
Beta-Lactam Antibiotic Resistance
itory concentration (MIC) of an antimicrobial agent,
or the least amount of antimicrobial agent that inhibits The earliest commercialized antibiotic, penicillin, was
the growth of an organism. Established guidelines define first used therapeutically in the early 1940s (Fig. 11.7).
the MICs interpreted as indications of susceptibility or Soon after that, resistance to penicillin through the
R O
O ONa
CH3 O
C O H
NH S N CH3
CH3 O
HN S CH3
O O N CH3 N
H S CH3 CH3 O
HC C C H
O
CH3 HO N CH3
C N CH O
–
COO
O FIGURE 11.9 Penicillin substitutes include methicillin (left)
FIGURE 11.7 Structure of penicillin, showing the carbon- and oxacillin (right).
nitrogen (CCCN) beta-lactam ring.
community. As described previously, expression of an
+
D + altered penicillin-binding protein (PBP2’ or PBP 2a
encoded by the mecA gene) is the resistance mechanism.
D N O NH
S
␤-Lactamase The antibiotics cannot bind to the altered target and
O N A –O2C
S therefore have no effect on the bacterial cells.
O OH– Rapid identification of MRSA isolates in clinical spec-
N A
CO2– imens by direct detection of mecA is critical for effective
H
CO2– patient management and prevention of infections occur-
ring in hospitals due to MRSA. PCR and other amplifi-
D cation assays have been developed for direct testing of
O NH clinical samples, and many assays have been tested for
+ sensitivity and specificity.
S A–
–O2C The stereochemical structure of the carbapenems
N makes them more resistant to inactivation by most
CO2–
plasmid and chromosomal-mediated β-lactamases than
the penicillins. After widespread use of these agents,
FIGURE 11.8 The reaction catalyzed by beta-lactamase however, resistance developed primarily through the
results in cleavage of the beta-lactam ring through an interme- production of carbapenem-hydrolyzing β-lactamases
diate product (bracketed). (carbapenemases).58
MALDI-TOF mass spectrometry peptide profiles
production of β-lactamases (Fig. 11.8) was recorded.
can discriminate resistant strains of organisms such as
Streptococcus pyogenes is one of the very few organ-
MRSA and VRE. In a mass spectrometry β-lactamase
isms that are still predictably susceptible to penicillin.
assay, the antibiotic is mixed with the bacterial culture.
Modified β-lactam molecules including the cephalo-
After incubation, the bacteria are removed by centrifuga-
sporins and carbapenems were subsequently developed.
tion, and the supernatant is analyzed for the hydrolyzed
Penicillin and the other β-lactam antimicrobials inhibit
form of β-lactam. This method can detect resistance to
bacteria by interfering with an enzyme that is involved
penicillin and a variety of β-lactam antibiotics, includ-
in the synthesis of the bacterial cell wall.
ing ampicillin, piperacillin, ceftazidime, imipenem, and
To counter the production of the bacterial
others.59 Modifications of the method have been aimed
β-lactamases (also known as penicillinases), penicillinase-
at increased speed and accuracy. Carbapenemase pro-
resistant penicillins—for example, methicillin or oxacillin
duction from anaerobic bacteria in less than 3 hours has
(Fig. 11.9)—were designed. Staphylococcal infections
been reported.60,61
were treated successfully with methicillin/oxacillin until
the emergence of resistance to these agents was first
Glycopeptide Antibiotic Resistance
observed in 1965.57 MRSA and methicillin-resistant
coagulase-negative staphylococci became a major cause Glycopeptide antibiotics were originally isolated from
of infections acquired in the hospital as well as in the plant and soil bacteria. Their structures contain either
HO The specificity of PCR and multiplex PCR was high

NH2
OH compared with the isolation of VRE in culture. Quan-
O
OH titative PCR has also been used to detect VRE in fecal
O surveillance specimens. Because PCR is faster than tra-
OH ditional culture methods and has comparable sensitiv-
OO ity and specificity to culture, it is an attractive method
Cl
O O for screening large numbers of samples for a particular
HO
6
Cl
4
OH
target.
O O
H H
O N N
N N NH Antimicrobial Resistance in M. tuberculosis
H H H
HN O O O N
O The use of molecular methods greatly improved antimi-
O– NH2
crobial resistance detection in M. tuberculosis because
OH
O OH traditional methods of determining antimicrobial suscep-
HO tibility took days, if not weeks, for this organism. The
FIGURE 11.10 Structure of vancomycin. longer a patient with tuberculosis (TB) is inadequately
treated, the more likely the development of resistance
and possible spread of the resistant organisms. Evolution
a glycosylated cyclic or polycyclic peptide. These anti- of drug resistance of M. tuberculosis in a noncompliant
biotics inhibit peptidoglycan synthesis in the cell wall patient for over 12 years was reported where subpopu-
of susceptible organisms (principally gram-positive lations of the organism emerged due to the acquisition
cocci). Glycopeptide antibiotics include vancomycin and accumulation of gene mutations that rendered the
(Fig. 11.10), teicoplanin, and ramoplanin; oritavancin, organism resistant to isoniazid, rifampin, and strepto-
dalbavancin, and telavancin are semisynthetic glyco- mycin.64 Many nucleic acid amplification protocols have
peptide antibiotics. Enterococcus was the first organism been developed to directly detect mutations in the genes
in which glycopeptide resistance was reported.62 Since associated with resistance to isoniazid and rifampin. In
then, vancomycin resistance has been observed in other general, these assays have demonstrated excellent sensi-
organisms. tivity and specificity and provide rapid determination of
The mechanism of vancomycin resistance can be drug susceptibility either directly from sputum or from
acquired or intrinsic to the organism. The antibiotic cultures.
binds and weakens cell wall structures of susceptible Thus, the advantages of using nucleic acid amplifi-
bacteria, ultimately causing cell lysis. Either the pres- cation assays for the determination of drug resistance
ence of the antibiotic itself or the weakening of the cell include the rapid and specific detection of mutations in
wall activates a set of van genes whose products modify genes associated with resistance to particular antimicro-
the cell wall structure such that the antibiotic no longer bial agents that provides irrefutable evidence of resis-
binds. The van genes are carried on a cassette localized tance in a short period.
to a plasmid or on the bacterial chromosome. Resistance
arises from enzymatic modifications of the aminogly-
cosides by acetyl-, adeno-, and phospho-transferases. MOLECULAR EPIDEMIOLOGY
A universal database for the detection of the modified
drugs by MALDI-TOF MS has been established. To An epidemic is a disease or condition that affects many
date, these methods are not yet in routine laboratory use unrelated individuals at the same time; a rapidly spread-
because they have not achieved the ease and reproducing outbreak of an infectious disease is an epidemic.
ibility of routine diagnostic systems.63 A pandemic is a disease that sweeps across wide geo-
PCR was used to detect the vanA, vanB, vanC1, graphical areas. Epidemiology includes collection and
and vanC2 resistance genes in fecal samples as a way analysis of environmental, microbiological, and clinical
to screen for vancomycin-resistant enterococci (VRE). data. In microbiology, studies are performed to follow
the spread of pathogenic organisms within a hospital sequencing methods. Mass spectrometry databases
(nosocomial infections), from the actions of a physician continue to accumulate for detailed typing of bac-
(iatrogenic infections), and in a community. Molecular teria and fungi by peptide profiles. The ability to
epidemiology is the study of causative genetic and envi- discern differences with increasing detail enhances the
ronmental factors at the molecular level to ascertain the capability to type organisms regardless of their com-
origin, distribution, and best strategies for prevention of plexity. All methods, however, have benefits and lim-
disease. In infectious disease, these efforts are facilitated itations with regard to instrumentation, methodology,
by the ability to determine the genetic similarities and and interpretation.
differences among microbiological isolates. Molecular methods are based on DNA and amino
In the laboratory, molecular methods are very useful acid sequences. Nucleotide and amino acid sequences
for identifying and typing infectious agents. In a single range from highly conserved across species and genera
patient, this is informative for therapeutic efficacy; in to unique to each organism. Some of these are strain-
groups of patients, it provides information for infec- or species-specific sequences but are still used for epi-
tion control. Typing systems are based on the premise demiological testing because these methods are highly
that clonally related isolates share molecular charac- reproducible and, depending on the targets, can dis-
teristics distinct from unrelated isolates. Molecular criminate between even closely related organisms. Most
technology provides analytical alternatives from the (but not all) molecular methods offer definitive results
chromosomal to the nucleotide sequence level. These in the form of DNA sequences, peptide profiles, or gel
genotypic methods, in addition to established pheno- band and peak patterns that can be interpreted objec-
typic methods, enhance the capability to identify and tively, which is less difficult than phenotypic determi-
type microorganisms. Whereas phenotypic methods are nations that often require experienced judgment.65 With
based on a range of biological characteristics, such as commercial systems, the performance has become rel-
antigenic type or growth requirements, genotypic pro- atively simple for some molecular epidemiology tests,
cedures target genomic or plasmid DNA (Table 11.11). whereas others require a higher level of laboratory
Genome scanning methods, such as restriction enzyme expertise.
analysis followed by PFGE, are used to find genetic
similarities and differences, as are amplification and
Molecular Strain Typing Methods
for Epidemiological Studies
In community or clinical settings, the same organism
TABLE 11.11 Epidemiological Typing Methods might be isolated multiple times, whether in the same
patient or from different patients. The physician has to
Class Methods determine whether collected isolates were independently
acquired, that is, came from different sources, or if they
Phenotypic Biotyping, growth on selective media came from the same source. With this knowledge, the
Antimicrobial susceptibility
Serotyping, immunoblotting actions are taken to control the transmission of the organ-
Bacteriophage typing ism, especially if it is being transmitted from a common
Protein, enzyme typing by electrophoresis source and that source has been identified. Most of the
MALDI-TOF mass spectrometry time, these analyses are performed on organisms that
Genotypic Plasmid analysis
have been transmitted nosocomially, but sometimes pro-
Restriction endonuclease mapping cedures to determine relatedness are performed on iso-
Pulsed-field gel electrophoresis lates from community outbreak situations.66
Ribotyping Many laboratory methods can be used to determine
Arbitrarily primed PCR, RAPD PCR the relatedness of multiple isolates, both phenotypic
Melt-curve analysis
REP-PCR, ERIC PCR, ITS, spa typing
(e.g., by MALDI-TOF, serology, and antimicrobial sus-
Mass parallel sequencing ceptibility patterns) and genotypic (e.g., PFGE and ribo-
typing). The phenotypic methods suffer from a lack of
reproducibility and their ability to discriminate between (0)

isolates. Even after recent advances in mass spectrom- 1 2 3 4 5 6 7
etry, genotypic methods are used almost exclusively to
type bacterial strains to determine the relatedness of
multiple isolates.
Plasmid Analysis
Plasmid analysis involves isolation and restriction
mapping of bacterial plasmids. The same bacterial strain
can have different plasmids carrying different pheno- 1 2 3 4 5 6 7
types or resistance patterns. For this analysis, plasmid
DNA is isolated from the specimen or culture and then
digested with restriction enzymes. Plasmids are distin-
guished by gel electrophoresis patterns of fragments
generated when cut with the appropriate enzymes.
Restriction analysis can also be performed on chro-
mosomal DNA of organisms with small genomes. For
organisms with larger genomes, whole genome analysis
with restriction enzymes that cut frequently enough to
identify plasmids will yield complex patterns that are
more difficult to interpret.
Pulsed-Field Gel Electrophoresis

Most molecular epidemiological tests are performed
using PFGE, which can identify organisms with larger
genomes or multiple chromosomes. For PFGE analysis,
the DNA is digested with restriction enzymes that cut
infrequently within the genomic sequences. The result-
ing large fragments (hundreds of thousands of base
pairs) are resolved by PFGE. Banding patterns will
differ depending on the chromosomal DNA sequence
FIGURE 11.11 PFGE of coagulase-negative Staphylococcus
of the organisms (Fig. 11.11). Tenover and colleagues
showing the outbreak (O) and four test strains (top panel). The
devised a system to interpret the banding pattern of a gel image is shown in the bottom panel. The fragment shifts
test organism compared to that of the strain of an iden- are marked in the top panel. Strains in lanes 4, 5, and 6 are the
tified or reference organism.67 The interpretation of same but different from the outbreak strain (lane 2). The strain
PFGE results follows the “rule of three” (Table 11.12), a in lane 3 is the same as the outbreak strain. The strains in lanes
method that has been used for typing numerous species, 4, 5, and 6 have at least five genetic differences and are not
including strains of Pseudomonas aeruginosa, Myco- related to the outbreak. Lanes 1 and 7, molecular-weight
bacterium avium, Escherichia coli, N. gonorrhoeae, markers. (Courtesy of Mary Hayden, MD, Rush Medical Laborato-
VRE, and MRSA. Intralaboratory and interlaboratory ries, Rush University Medical Center, Chicago, IL.)
computerized databases of band patterns can be stored
for reference. A national PFGE database is stored at
Restriction Fragment Length
the CDC (www.cdc.gov/pulsenet). One disadvantage
Polymorphism Analysis
of using PFGE to type strains is the time involved to
perform the assay; it can take 2 to 3 days to complete RFLP analysis by Southern blot is the same technique
one analysis. first used to identify and investigate human genes. This
TABLE 11.12 Criteria for PFGE Pattern Interpretation
Category Genetic Differences* Fragment Differences* Epidemiological Interpretation
Indistinguishable 0 0 Test isolate is the same strain as the outbreak strain
Closely related 1 2–3 Test isolate is closely related to the outbreak strain
Possibly related 2 4–6 Test isolate is possibly related to the outbreak strain
Different ≥3 ≥6 Test isolate is unrelated to the outbreak
*Compared with the outbreak strain.
method involves cutting DNA with restriction enzymes, much easier to interpret. Although the method is limited
resolving the resulting fragments by gel electrophoresis, by the sequences that can be amplified and differenti-
and then transferring the separated fragments to a mem- ated through restriction enzyme digestion, proper gene
brane for probing with a specific probe. Gene-specific selection provides a highly reproducible and discrimi-
probes are used to identify or subtype microorganisms natory test. In one study, an 820-bp amplified fragment
such as P. aeruginosa in patients with cystic fibrosis and of the ureC gene from Helicobacter pylori digested with
nosocomial L. pneumophila infections. Sau3A and Hhal yielded 14 different Sau3A patterns and
For strain typing of M. tuberculosis by RFLP, the 15 different Hhal patterns. These patterns were informa-
probe is complementary to an insertion element, IS6110, tive as to the antibiotic sensitivity of the various types to
and will bind to restriction fragments on the membrane clarithromycin therapy.
that contains it, resulting in a series of bands that are
easily analyzed and compared. Insertion elements (inser- Arbitrarily Primed PCR
tion sequences) are segments of DNA that move inde-
Arbitrarily primed PCR, or random amplified poly-
pendently throughout the genome and insert themselves
morphic DNA (RAPD) assay, is a modified PCR using
in multiple locations. Strains can be RFLP-typed based
short (e.g., 10-base-long) oligonucleotides of random
on how many insertions are present and where they are
sequences to prime DNA amplification all over the
located. Isolates from the same strain will have the same
genome. The gel pattern of amplicons produced is char-
number and location of elements.
acteristic of a given organism. If two organisms have the
The gene targets selected for RFLP depend on the
same pattern, they are considered the same type; if the
organism under investigation and which genes will be
patterns differ, they are different types. The RAPD assay
most informative. Ribosomal RNA genes are highly
is relatively fast and inexpensive; however, producing
informative over a range of microorganisms, which is
consistent results may be technically demanding. Accu-
the basis for a modification of the RFLP procedure,
rate interpretation of RAPD raw data requires that the
which is a form of ribotyping. For this method, probes
procedure conditions be followed strictly so that pattern
target the 16S and 23S rRNA genes. RFLP and ribotyp-
differences (not necessarily patterns) are reproducible
ing have been applied in industrial as well as clinical
(Fig. 11.12).
microbiology.
RFLP can be performed more rapidly using PCR
Amplified Fragment Length Polymorphism
amplification with gene-specific primers (locus-specific
(AFLP) Assay
RFLP [PCR-RFLP]). This method requires the ampli-
fication of specific regions by PCR. The amplicons are AFLP is a name, rather than an acronym, chosen by the
then cut with restriction enzymes, yielding bands of in- inventors of this assay due to its resemblance to RFLP.
formative size. An advantage of this procedure, in addi- The AFLP assay is based on the amplification of DNA
tion to its speed, is the simple banding pattern, which is fragments generated by cutting the test genome with
M C
A
M C
FIGURE 11.12 RAPD gel results. An unacceptable

gel pattern is represented in panel A. The bands are
smeared, and variable producing patterns are too
complex for positive identification of unrelated
strains. The gel pattern represented in panel B is
acceptable. Strain differences can be clearly identi-
fied by variations from the known strain (C). Molec-
ular-weight markers are shown in lane M. B
restriction enzymes. DNA isolated from the test strain transposable elements (stretches of DNA that move from
is digested with HindIII or other restriction enzyme one location to another in a non-Mendelian fashion; also
(Fig. 11.13). Short DNA fragments or adaptors are called jumping genes). The genomic locations of these
ligated to the ends of the cut DNA. The adaptor-ligated structures are related to species type and can be used to
fragments are then amplified in two steps with primers distinguish between bacterial isolates.
complementary to the adaptor sequences. Nucleotides Enterobacterial repetitive intergenic consensus
located at the 3′ end of the primers select for specific (ERIC) sequences are 126-bp-long genomic sequences
sequences in the restriction fragments. The amplicons found in some bacterial species that are highly con-
are then resolved by gel or capillary electrophoresis (flu- served, even though they are not in coding regions.
orescently labeled primers are used for capillary electro- These sequences are located between genes in operons
phoresis). The pattern will be characteristic of the strain or upstream or downstream of single open reading
or type of organism. This assay can be performed with frames. ERIC sequences are flanked by inverted repeats
one or two enzymes (e.g., EcoR1/MseI or BamH1/PstI). that could form stem-loop or cruciform structures in
AFLP may detect more polymorphisms than RAPD DNA and are found only in gram-negative organisms,
analysis and is faster than PFGE. The procedure is more such as Bartonella, Shigella, Pseudomonas, Salmonella,
technically demanding, however, than a similar method, Enterobacter, and others.
REP-PCR (discussed in the next section). Gel patterns A related type of repetitive element, the repetitive
may also be complex (Fig. 11.14). Both high and low extragenic palindromic (REP) sequence, is similar to the
reproducibility for the method have been reported. ERIC sequence in that it occurs in noncoding regions
and contains an inverted repeat. REP sequences differ
from ERIC sequences in size (REP sequences are only
Interspersed Repetitive Elements
38 bp long), in being more numerous in the genome, and
Copies of conserved sequences are found through- in being present in multiple copies at a single location
out the genomes of most organisms. These sequences (Fig. 11.15). PCR primed from these elements yields a
may have arisen from viral integration or movement of series of products that can be resolved by gel, capillary
Chromosomal DNA
HindIII fragment
5′ AAG CTT A 3′
3′ A TTC G A A 5′
Ligate adaptors
Adaptor Adaptor
5′ AAG CTT AAGCTT 3′
3′ TTCG A A TTC G A A 5′
Amplify with
preselective primers FIGURE 11.13 AFLP analysis begins with
restriction digestion of chromosomal DNA.
N
The resulting fragments (top) are ligated with
5′ AAG CTT AAGCTT 3′ adaptors compatible with the restriction
3′ TTCG A A TTC G A A 5′ enzyme ends and complementary to primers
N used to amplify them. The first amplification is
Amplify with performed with preselective primers that end
selective primers
in a 3′ base (N) selected by the user. Selective
NNN primers with three added 3′ bases are used for
5′ AAG CTT AAGCTT 3′ a second round of PCR. This selection results
3′ TTCG A A TTC G A A 5′ in a characteristic pattern; only a fraction of
the original fragments will be represented in
NNN
the gel pattern.
electrophoresis, or microfluidics into characteristic pat- is located between the 5.8S and the 28S rRNA genes.
terns (Fig. 11.16). These elements have been used for Two additional elements, intergenic spacer (IGS)
typing of clinically important organisms, such as Clos- regions, IGSI and IGSII, are located between the
tridium difficile and fungal pathogens. rDNA repeat units (Fig. 11.17). Used for the identifi-
Another repetitive element, BOX, was discovered in cation and typing of yeast and molds, ITS sequences
S. pneumoniae. BOX elements consist of different com- are conserved within species but polymorphic between
binations of subunits, boxA, boxB, and boxC, which species. The ITS sequences are amplified using primers
are 59, 45, and 50 bp long, respectively. Although these directed to the unique 17S and 26S gene sequences. The
elements are not related to ERIC and REP sequences, resulting amplicons are analyzed by sequencing, sin-
they do form stem-loop structures, as do ERIC and REP. gle-strand conformation polymorphism, density-gradi-
About 25 of these elements are present in the S. pneumo- ent gel electrophoresis, restriction enzyme analysis, or
niae genome, where they may be involved in regulation sequence-specific PCR.
of gene expression.
spa Typing
Internal Transcribed Spacer Elements
MRSA contains a VNTR element in the 3′ coding region
The ribosomal RNA genes comprise the most con- of the protein A gene (spa). The element consists of repeat
served region in the genome. These genes are arranged units of 21 or 24 bp in length. Repeat units also vary by
as an operon, including a small subunit, 18S rRNA, sequence of at least one position. The spa element can
5.8S rRNA, and a large subunit, 28S rRNA. The ITS 1 have 2 to 16 repeat units. Analysis of these elements by
and 2 elements (ITS1 and ITS2) are found in regions PFGE or sequencing and comparison to known isolates
separating the 18S and the 28S rRNA genes. ITS1 is (spa typing) is used to identify MRSA. Almost 400
located between the 18S and the 5.8S gene, and ITS2 repeat profiles or spa types have been defined.
A similar sequence structure in the coagulase this method is increased by analysis of other repeated
gene (coa) has also been used for S. aureus typing sequences elsewhere in the MRSA genome. The com-
(coa typing). The coa VNTR units are 81 bp in length. bination of spa and VNTR typing has a discrimina-
PCR amplification and sequence analysis are used tory power equal to that of PFGE, with a more rapid
to analyze coa types. The discriminatory power of turnaround time.
1 1 2 2 3 3 Isolate A
Isolate B
M A B M A B U
FIGURE 11.16 Species identification by REP or ERIC

primed amplification. REP and ERIC sequences are present in
different chromosomal locations in bacterial subtypes A and B.
PCR, which extends outward-oriented primers hybridized to
the repeated sequences, generates amplicons of different sizes
based on the placement of the element, as in the gel depicted
in the bottom left panel where only one amplicon is shown.
FIGURE 11.14 Banding patterns generated by fluorescent Multiple REPs and ERICs throughout the bacterial chromo-
AFLP analysis. Note that duplicate specimens (1, 2, and 3) do some generate multiple amplicons with characteristic gel pat-
not produce the exact pattern because of band shifts and differ- terns (bottom right). An unknown isolate (U) is identified as
ent band intensities. the same strain as isolate B. M, molecular-weight markers.
ERIC sequence
…GTGAATCCCCAGGAGCTTACATAAGTAAGTGACTGGGGTGAGCG…
REP sequence
…GCC G/T GATGNCG G/A CG C/T NNNNN G/A CG C/T CTTATC C/A GGCCTAC…
FIGURE 11.15 The central inverted repeat of a 126-bp ERIC sequence (top) and a consensus REP sequence (bottom). ERIC
sequences contain multiple inverted repeats (arrows) that can generate a secondary structure consisting of several stems and loops.
REP sequences contain a conserved inverted repeat that forms a stem with a loop that includes 5 bp of variable sequence (N).
One rRNA repeat unit

rRNA
SSU (18S) ITS1 5.8S ITS2 LSU (28S) IGS1 5S IGS2 SSU (18S) ITS1 5.8S ITS2 LSU (28S) IGS1 5S
FIGURE 11.17 Ribosomal RNA genes are arranged in multiple tandem units that include the major rRNA transcript (18S to 28S)
and the 5S gene. ITSs are located within the major transcript template area and IGSs in the region between the repeat units sur-
rounding the 5S rRNA gene.
MALDI-TOF peak patterns or peptide mass profiles.68

TABLE 11.13 Example of Housekeeping Genes The profiles are analyzed by searching for matching
Sequenced in an MLST Test profiles in extensive open-source and intralaboratory
databases. MALDI methods are faster than some genetic
Number methods and can successfully detect organisms from
Gene Gene Product of Alleles
mixtures. Use of MALDI results for stain comparisons
ArcC Carbamate kinase 52 is limited, however. Peptide profiles are influenced by
growth conditions which affect microbial physiol-
AroE Shikimate dehydrogenase 88 ogy and protein expression. Although identification by
GlpF Glycerol kinase 55 mass spectrometry requires culture of microorganisms,
culture conditions and time of culture do not seem to
Gmk Guanylate kinase 51 alter profiles.69,70
Pta Phosphate acetyltransferase 57 Speciation of organisms that cause environmen-
tal hazards, such as food- and water-borne diseases, is
Tpi Triosephosphate isomerase 74 important not only for investigating but also for prevent-
YqiL Acetyl coenzyme A acetyltransferase 66 ing outbreaks. Only some strains of organisms are impli-
cated in outbreaks. Peptide maps of organisms, such as
Aeromonas, from which 7 of 17 species are related to
Multilocus Sequence Typing water outbreaks, allow accurate classification of species
for environmental monitoring.71 MALDI has also been
Multilocus sequence typing (MLST) characterizes bac- applied to the identification of food pathogens such as
terial isolates by using sequences of internal fragments lactic acid bacteria from spoiled food, toxin-producing
of housekeeping genes. Six or seven genes are sequenced bacteria responsible for food poisoning, and beneficial
over 450 to 500 bases, and the sequences are assigned bacteria in probiotics and yogurt.
as alleles. Examples of housekeeping genes used in Whole-cell MALDI-TOF mass spectrometry can be
S. aureus MLST are shown in Table 11.13. Distinct used to identify and compare organisms from highly
alleles are defined as single-base-pair differences or contaminated ecosystems, such as soil and sewage
multiple changes resulting from recombination or other sludge.72 In this application, intact cells are extracted
genetic events. An isolate type or the allelic profile is the with trifluoroacetic acid and acetonitrile and permeabi-
collection of all seven alleles, also called the sequence lized with glass beads before ionization. This method is
type. Bacteria have enough variation so that there are also applied to intact conidia and spores for identifica-
multiple alleles of each housekeeping gene, making up tion of fungal organisms.
billions of allelic profiles. Because the data generated by
MLST is text sequence, the results can be compared with
those in large databases, for example, http://pubmlst.org. Comparison of Typing Methods
Genotypic methods used for strain typing are evaluated
Mass Spectrometry
and compared based on five criteria. First, the target
Peptides (2 to 20 kd) of the highly abundant ribo- organism must be typable by the method used to detect
somal proteins are assessed to identify microbes by it (typing capacity). A test that detects a genotypic or
TABLE 11.14 Performance Comparison of Representative Molecular Epidemiology Methods*
Typing Discriminatory Ease of

Method Capacity Power Reproducibility† Ease of Use Interpretation‡
Plasmid analysis Good Good Good High Good
PFGE High High High Moderate Good–Moderate
Chromosomal RFLP High Good Good High Moderate–Poor
Ribotyping High High High Good High
PCR-RFLP Good Moderate Good High High
RAPD High High Poor High Good–High
AFLP High High Good Moderate High
Repetitive elements Good Good High High High
Sequencing High High High Moderate Good–High
Mass spectrometry High Good-High Good Good High
*From Olive and Bean.76

†
Intralaboratory
‡
Interpretation is influenced by the quality of the data.
phenotypic characteristic that is not expressed in all interpretation. Unfortunately, no such method fits this
members of a species will not accurately detect the profile, so the laboratory professionals performing these
target organisms at all times. A molecular assay must types of analyses may have to sacrifice ease of perfor-
target a reasonably polymorphic DNA sequence, alleles mance, for example, in order to get excellent discrimi-
of which are unambiguously associated with a given natory power when they are choosing which molecular
strain. Second, the method must be reproducible. A typing method to use.
reproducible method yields the same result on repeated In conclusion, molecular-based methods are available
testing of the same organism or strain. Variations in cell for the detection, identification, and characterization of
characteristics, such as antigens or receptor expression, a number of microorganisms. For some, assays are used
decrease reproducibility (precision). Third, the method almost exclusively, such as for N. gonorrhoeae, C. tra-
must clearly distinguish between unrelated strains (dis- chomatis, and B. pertussis. For others, molecular-based
criminatory power). Reproducibility and discriminatory tests are used to provide rapid results and supplement
power are important for the establishment of databases traditional testing, such as for M. tuberculosis. Finally,
that can be used by independent laboratories. Fourth, molecular-based methods are undergoing continuous
ease of interpretation of results is important. Unclear development and experiencing increasing use compared
or complex results will lower reproducibility and dis- to traditional culture and phenotypic methods.
criminatory power. Finally, ease of test performance is
important in order to minimize the chance of error or
ambiguous results. A rating of representative methods is Case Study 11.1
shown in Table 11.14.
The most desirable typing method is the one that During a holiday weekend at a luxury hotel, guests
will type all strains and have excellent reproducibil- began to complain of stomach flu with nausea and
ity, discriminatory power, and ease of performance and
vomiting. In all, more than 100 of the 200 guests Case Study 11.2
who had dined at the hotel the previous evening
described the same symptoms. Eight people had Five students from different local community
symptoms severe enough to warrant hospital- high schools suffered recurrent skin infections
ization. Most, however, recovered within 24 to with chronic wounds. Nasal swabs and skin
48 hours of the onset of symptoms. Health offi- specimens from the students were screened for
cials were notified. Interviews and epidemiological MRSA by inoculation onto Mueller–Hinton agar
analyses pointed to a Norwalk-like virus infection, supplemented with NaCl (0.68 mol/L) containing
or norovirus, probably foodborne. Stool specimens 6 μg/mL of oxacillin. The cultured organisms
(1 to 5 mL) from hospitalized patients and samples exhibited an MIC of more than 32 μg/mL vanco-
of the suspected food sources (500 mg) were sent mycin and a zone of inhibition of less than 14 mm
for laboratory analysis by RT-PCR. RNA extracted in diameter. Isolates were sent to the CDC and
with 1,1,2 trichloro-1,2,2, trifluoroethane was referred for molecular testing. DNA isolated from
mixed with a guanidium thiocyanate buffer and iso- the five cases and a control strain were embedded
lated by organic (phenol-chloroform) procedures. in agarose plugs and digested with SmaI for PFGE
cDNA was synthesized using primers specific to analysis. A depiction of the gel pattern is shown in
the viral RNA polymerase gene, and strain-specific the accompanying figure.
PCR primers were used to amplify the viral gene. M 1 2 3 4 5 6 7 M
The amplicons, resolved by agarose gel electro-
phoresis, are shown in the accompanying figure.
PFGE patterns of isolated strains. M, molecular-weight

markers. Lanes 1 to 5, isolates from the community infections;
lanes 6 and 7, unrelated hospital isolates.
RT-PCR products were resolved on separate gels. Amplicons Further PCR testing was performed on the iso-
from four affected individuals (lanes 1 to 4, left). Lane 5, posi- lates for virulence factors, particularly for mecA
tive control; lane 6, sensitivity control; lane 7, negative control;
lane 8, reagent blank. Specimens from suspected food sources
gene sequencing and detection of the Panton–
(lanes 1 to 4, right). Lane 5, salad lettuce from the distributor; Valentine leukocidin (PVL) genes, lukS-PV and
lanes 6 to 8, specimens from three hotel employees working lukF-PV. The results from these tests revealed that
the day of the outbreak; lane 9, molecular-weight marker. all five isolates contained the PVL genes and the
type IV mecA element.
QUESTIONS:
QUESTIONS:
1. Do the four patients have norovirus?
1. Are all or some of the five isolates the same
2. What is the source of the organism?
or different? Which isolates are the same, and
3. When did the source get contaminated? Did the
which are different? What is the evidence to
source come into the hotel infected, or was it
support your answer?
infected inside the hotel?
2. Was there a single source for these organisms
or multiple sources?
c. Ribotyping
Case Study 11.3 d. Bacteriophage typing
A 39-year-old HIV-positive male has been moni- 3. For which of the following organisms must caution
tored closely since his diagnosis as HIV-positive be exercised when evaluating positive PCR results
5 years ago. The man was on HAART and com- because the organism can be found as normal flora
pliant. He was relatively healthy and had not even in some patient populations?
had a cold in the last 4 years. The man had HIV
viral loads as determined by Amplicor RT-PCR a. Neisseria gonorrhoeae
that were consistently close to 10,000 copies/mL, b. HIV
never varying more than 0.2 log10 unit, until the c. Chlamydophila pneumoniae
last 6 months, when his viral loads were trending d. Streptococcus pneumoniae
up to 25,500, then 48,900, with his most recent
result being 55,000 copies/mL. Genotyping per- 4. Which of the following controls are critical for
formed on virus isolated from the patient revealed ensuring that amplification is occurring in a patient
a mutation in the reverse transcriptase gene of sample and that the lack of PCR product is not due
M41L that is associated with resistance to zidovu- to the presence of polymerase inhibitors?
dine (AZT). a. Reagent blank
QUESTIONS: b. Sensitivity control
1. What is the significance of the viral load results c. Negative control
over the last 6 months? d. Amplification control
2. What is the implication of the genotyping result
for the patient's therapy? 5. A PCR assay was performed to detect Bordetella
3. How should this patient be monitored in the pertussis on sputum obtained from a 14-year-old
future? girl who has had a chronic cough. The results
revealed two bands, one consistent with the
internal control and the other consistent with
the size expected for amplification of the
B. pertussis target. How should these results
be interpreted?
STUDY QUESTIONS a. These are false-positive results for B. pertussis.
b. The girl has clinically significant B. pertussis
infection.
1. Which of the following genes would be analyzed
c. B. pertussis detection is more likely due to
to determine whether an isolate of Staphylococcus
colonization.
aureus is resistant to oxacillin?
d. The results are invalid because two bands were
a. mecA present.
b. gyrA
c. inhA 6. Which of the following is an advantage of
d. vanA molecular-based testing?
a. Results stay positive long after successful
2. Which of the following is a genotypic method
treatment.
used to compare two isolates in an epidemiological
b. Results are available within hours.
investigation?
c. Only viable cells yield positive results.
a. Biotyping d. Several milliliters of specimen must be
b. Serotyping submitted for analysis.
7. Which molecular-based typing method has high universal viral transport medium during an epidemic. Journal of
typing capacity, reproducibility and discriminatory Clinical Microbiology 2014;52:2656–2658.
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Chapter 12
Molecular Detection
of Inherited Diseases
Outline Objectives
THE MOLECULAR BASIS OF INHERITED DISEASES 12.1 Describe Mendelian patterns of inheritance as
CHROMOSOMAL ABNORMALITIES exhibited by family pedigrees.
PATTERNS OF INHERITANCE IN SINGLE-GENE DISORDERS 12.2 Illustrate abnormalities in chromosome number
MOLECULAR BASIS OF SINGLE-GENE DISORDERS and structure.
Lysosomal Storage Diseases 12.3 Define penetrance and variable expressivity.
Factor V Leiden 12.4 Relate disease syndromes with affected genes.
Prothrombin 12.5 Give examples of laboratory methods designed to
Methylenetetrahydrofolate Reductase detect single-gene disorders.
Hemochromatosis 12.6 Discuss non-Mendelian inheritance, and give
Cystic Fibrosis examples of these types of inheritance, such as
Cytochrome P-450 mitochondrial disorders and nucleotide-repeat
SINGLE-GENE DISORDERS WITH NONCLASSICAL PATTERNS expansion diseases.
OF INHERITANCE 12.7 Show how genomic imprinting can affect disease
Mutations in Mitochondrial Genes phenotype.
Nucleotide-Repeat Expansion Disorders
Fragile X Syndrome
Huntington Disease
Idiopathic Congenital Central Hypoventilation Syndrome
Other Nucleotide Expansion Disorders
Genomic Imprinting
Multifactorial Inheritance
LIMITATIONS OF MOLECULAR TESTING
344
Chapter 12 • Molecular Detection of Inherited Diseases 345
Genetic and cytogenetic analyses are a critical com-

ponent of diagnostic testing, especially for diseases Advanced Concepts
that arise from known genetic events. The identifica-
tion of a molecular or chromosomal abnormality is a According to the Lyon hypothesis (or Lyon’s
direct observation of the source of some diseases. This hypothesis) first stated by Mary Lyon in 1961,
chapter presents examples of clinical laboratory tests only one X chromosome remains genetically
commonly performed in molecular genetics using these active in females.1,2 In humans, one X chromo-
techniques. some is inactivated at random about the 16th day
of embryonic development. The inactive X can be
seen as a Barr body (X chromatin) in the inter-
THE MOLECULAR BASIS phase nucleus. Not all X genes are shut off in the
OF INHERITED DISEASES inactivated X chromosome. Furthermore, reactiva-
tion of genes on the inactivated X occurs in germ
Mutations are changes in DNA nucleotide sequences. cells before the first meiotic division for produc-
These changes range from single base pair or point tion of eggs.
mutations of various types to chromosomal. Not all
mutations lead to disease.
Polymorphisms are proportionately represented gen-
otypes in a given population. Sequence polymorphisms
can be located within genes or outside of genes. Benign CHROMOSOMAL ABNORMALITIES
polymorphisms are useful for mapping disease genes,
determining parentage, and identity testing. Balanced Genome mutations (abnormalities in chromosome
polymorphisms can have offsetting phenotypes. number) can be detected by karyotyping, ploidy analysis
Epigenetic alterations do not change the primary by flow cytometry, and fluorescent in situ hybridization
DNA sequence. Epigenetic changes consist of three (FISH). Polyploidy (more than two of any autosome)
different forms: DNA methylation, usually alterations in animals usually results in infertility and abnormal
of cytosine in CpG islands; genomic imprinting; and appearance. Aneuploidy (gain or loss of any autosome)
chromatin remodeling. DNA methylation mostly down- occurs with 0.5% frequency in term pregnancies and
regulates RNA transcription. Genomic imprinting 50% in spontaneous abortions. Aneuploidy is caused
selectively inactivates chromosomal regions (e.g., X chro- by erroneous separation of chromosomes during egg or
mosome inactivation). Chromatin remodeling sequesters sperm production (chromosomal non-disjunction). Auto-
large regions of chromosomal DNA through protein somal trisomy/monosomy (three copies/one copy of a
binding and histone modification. Histone modification chromosome instead of two) results from fertilization
controls the availability of DNA for RNA transcription. of gametes containing an extra chromosome or missing
Mutations in germ cells result in inherited disease. a chromosome (n + 1 or n – 1 gametes, respectively).
Mutations in somatic cells result in cancer and some Autosomal monosomy is generally, but not always,
congenital malformations. Diseases with genetic compo- incompatible with life. Sex chromosome aneuploidy is
nents are often referred to as congenital (“born with”) more frequently tolerated, although it is associated with
diseases. Congenital disorders are not necessarily her- phenotypic abnormalities.
itable, however. Congenital disorders are those present Mosaicism, two or more genetically distinct pop-
in individuals at birth. Specifically, congenital disorders ulations of cells from one zygote in an individual (in
result when some factor, such as a drug, a chemical, an contrast to chimerism: two or more genetically distinct
infection, or an injury, upsets the developmental process. cell populations from different zygotes in an individual),
Thus, a baby can have a heritable disease, such as hemo- results from mutation events affecting somatic or germ
philia, that can be passed on to future generations or a cells. Early segregation errors during fertilized egg divi-
congenital condition, such as spina bifida, that cannot be sion occasionally give rise to mosaicism. Mosaicism is
passed to offspring. relatively common with sex chromosomes; for example,
TABLE 12.1 Examples of Genome Mutations
Genetic
Disorder Abnormality Incidence Clinical Features
Down syndrome Trisomy 21, 47,XY,+21 1/700 live births Flat facial profile, mental retardation, cardiac problems,
risk of acute leukemia, eventual neuropathological
disorders, abnormal immune system
Edward syndrome Trisomy 18, 47,XY,+18 1/3,000 live births Severe, clenched fist; survival less than 1 year
Patau syndrome Trisomy 13, 47,XY,+13 1/5,000 live births Cleft palate, heart damage, mental retardation, survival
usually less than 6 mo
Klinefelter syndrome 47,XXY 1/850 live births Male hypogonadism, long legs, gynecomastia (male
breast enlargement), low testosterone level
XYY syndrome 47,XYY 1/1,000 live births Excessive height, acne, 1%–2% behavioral disorders
Turner syndrome 45,X and variants 1/2,000 live births Bilateral neck webbing, heart disease, failure to develop
secondary sex characteristics, hypothyroidism
Multi X females 47,XXX; 48,XXXX 1/1,200 newborn Mental retardation increases with increasing X
females
45,X/47,XXX (normal female chromosome complement somatic events (not inherited) and are most commonly
is 46,XX). In this case, later nondisjunction will yield seen in cancer. Approximately 7.4% of conceptions
additional populations. Rarely, autosomal haploids will have chromosome mutations. Chromosome mutations
be lost with the retention of the triploid lineage (e.g., are observed in 50% of spontaneous abortions and 5%
45,XY,-21, 46,XY/47,XY,+21 → 46,XY/47,XY,+21). of stillbirths. Examples of diseases arising from inher-
Examples of genome mutations are shown in Table 12.1 ited chromosome structure abnormalities are shown in
Chromosome mutations (abnormalities in chromosome Table 12.2.
structure) larger than 4 million base pairs (bp) can be
seen by karyotyping; smaller irregularities can be seen
with the higher resolution of FISH or microarray tech- PATTERNS OF INHERITANCE
nology. Structural alterations include translocations IN SINGLE-GENE DISORDERS
(reciprocal, nonreciprocal), inversions (paracentric, peri-
centric), deletions (terminal, interstitial, ring), duplica- Most phenotypes result from the interaction of multiple
tions (isochromosomes), marker chromosomes, and genetic and environmental factors. Some phenotypes,
derivative chromosomes. however, are caused by alteration of a single gene. If the
Structural mutations require breakage and reunion of phenotype occurs as predicted by Mendelian genetics,
DNA. Chromosomal breakage is caused by chemicals patterns of inheritance can be established. Patterns of
and radiation. Chromosomal breakage also results from inheritance (transmission patterns) are determined by
chromosome breakage syndromes (e.g., Fanconi anemia, examination of family histories. A pedigree is a diagram
Bloom syndrome, and ataxia telangiectasia). Some aber- of the inheritance pattern of a phenotype of family
rations have no immediate phenotypic effect (recip- members (Fig 12.1). There are three main Mendelian
rocal translocations, inversions, some deletions, some transmission patterns: autosomal dominant, autosomal
insertions). Others can be deleterious to cells includ- recessive, and X-linked or sex-linked recessive. These
ing lethality. Chromosome translocations are usually patterns refer to the disease phenotype.
TABLE 12.2 Examples of Chromosomal Mutations
Genetic
Disorder Abnormality Incidence Clinical Features
DiGeorge syndrome and del(22q) 1/4,000 live births CATCH 22 (cardiac abnormality/abnormal facies,
velocardiofacial syndrome T-cell deficit, cleft palate, hypercalcemia)
Cri du chat syndrome del(5p) 1/20,000–1/50,000 Growth deficiency, catlike cry in infancy, small
live births head, mental retardation
Contiguous gene syndrome; Wilms’ del(11p) 1/15,000 live births Aniridia (absence of iris), hemihypertrophy (one
tumor, aniridia, genitourinary side of the body seems to grow faster than the
anomalies, mental retardation other), and other congenital anomalies
syndrome
FIGURE 12.2 In an autosomal-dominant transmission

pattern, heterozygous individuals express the affected pheno-
type (filled symbols).
Male Female
Deceased male Deceased female that Mendelian inheritance is still present and that the
Affected male Affected female partial dominance is a manifestation of how the genes
are expressed. Codominant offspring simultaneously
FIGURE 12.1 A pedigree is a diagram of family phenotype demonstrate the phenotype of both parents. A familiar
or genotype. The pedigree will display the transmission pattern example of codominance is the ABO blood types. Dom-
of a disease. Phenotypic traits are followed by coloring or pat- inant-negative phenotypes are seen in cases of multim-
terns in the symbols. Lack of the trait is indicated by open eric proteins such as the tumor-suppressor tetramer, p53
symbols with no coloring.
(Fig. 12.3). Even though only one allele is mutated, the
mutated protein can interfere with the function of the
In autosomal-dominant transmission, a child of an tetramer, producing an abnormal phenotype.
affected individual and an unaffected mate has a 50% to The phenotype of a loss-of-function mutation is
100% risk or likelihood of expressing the disease pheno- usually recessive, but it depends on the type of protein
type (Fig. 12.2). Gain-of-function mutations are usually affected. Complex metabolic pathways are suscepti-
dominant because the mutated allele produces sufficient ble to loss-of-function mutations because of extensive
abnormal factor to bring about the affected condition. interactions between and among proteins. Key structural
In complete dominance, the heterozygous phenotype proteins, especially multimeric complexes, risk domi-
of the offspring is the same as that of the homozygous nant negative phenotypes. Gain-of-function mutations
parent. In partial dominance, the offspring phenotype are less common than loss-of-function mutations. Gain-
is variably intermediate between the homozygous and of-function mutations include gene-expression/stability
heterozygous parental phenotypes. The parental phe- defects that generate gene products at inappropriate sites
notypes can reappear in the next generation, showing or times.
Autosomal recessive is the largest category of Men- mutations) such as type 2 diabetes may also be inborn
delian disorders. The recurrence risk is 25% if siblings errors of metabolism.3
are affected, indicating the presence of the recessive
mutation in both of the parents. The “margin of safety,” Advanced Concepts
that is, having two copies of every gene, requires the
loss of the normal allele through somatic events (loss of Autosomal-dominant mutations can originate from
heterozygosity) or homozygosity for the manifestation gonadal mosaicism of new mutations in germ
of the recessive disease phenotype. Autosomal-recessive cells, that is, DNA changes that arise in cells that
diseases are more often observed as a result of two indi- produce eggs or sperm. Establishment of a new
viduals heterozygous for the same mutation producing mutation as a dominant mutation in a family or in
offspring (Fig. 12.4). New mutations are rarely detected a population is influenced by its effect on repro-
in autosomal-recessive transmission patterns. Inborn ductive fitness.
errors of metabolism are usually autosomal recessive.
Risk factors for neoplastic diseases also fall in this cat-
egory. Polygenic disorders (caused by multiple gene Almost all sex-linked disorders are X-linked because
relatively few genes are carried on the Y chromosome.
Chromosomes X-linked mutations are almost always recessive, but
there are X-linked dominant diseases (e.g., vitamin
Monomers Tetramers D–resistant rickets). Even though one X chromosome
is inactivated in females, the inactivation is reversible
+ +
so that a second copy of X-linked genes is available.
+ Normal
function
In contrast, males are hemizygous for X-linked genes,
+ having only one copy on the X chromosome. Males,
+ + therefore, are more likely to manifest the disease phe-
notype (Fig. 12.5).
+ +
+
Abnormal
+
– –
FIGURE 12.4 Autosomal-recessive mutations are not

FIGURE 12.3 Dominant negative mutations affect multim- expressed in heterozygotes. The phenotype is displayed only in
eric proteins. In this illustration, a single mutant monomer a homozygous individual; in this illustration, produced by the
affects the function of the assembled tetramer. inbreeding of two cousins (double horizontal line).
FIGURE 12.5 X-linked recessive diseases are carried

by females but manifested most often in males.
Due to the multifactorial nature of most diseases, substrates. With the discovery of genes that code for the
the same gene mutations are not always manifested in enzymes and their subunits, molecular testing has been
a disease phenotype. Penetrance is the frequency of used to some extent. Mutations can be detected by direct
expression of disease phenotype in individuals with a sequencing, usually after an initial biochemical screen-
gene lesion. Complete penetrance is the expression of the ing test for loss of enzyme activity.
disease phenotype in every individual with the mutated
gene. Complete penetrance is common in homozygous
Factor V Leiden
recessive phenotypes. Variable expressivity is a range
of phenotypes in individuals with the same gene lesion. Mutations that lead to abnormal but survival states can
Variable expressivity also reflects the interaction of be relatively frequently encountered in a population. An
other gene products and the environment on the disease example is the hypercoagulation phenotype resulting in
phenotype. mutations in the factor V gene. Discovered in 1994, the
Leiden mutation (1691 A→G, R506Q) in the coagula-
tion factor V gene F5 (1q23) causes a hypercoagulable
MOLECULAR BASIS (thrombophilic) phenotype. This genotype is present in
OF SINGLE-GENE DISORDERS heterozygous form in 4% to 8% of the general popu-
lation, and 0.06% to 0.25% of the population is homo-
Single-gene disorders affect structural proteins, cell zygous for this mutation. A blood clot or deep venous
surface receptor proteins, growth regulators, and thrombosis is treated with anticoagulants. The risk of
enzymes. Examples of diseases resulting from such dis- thrombosis increases with contraceptive use in women
orders are shown in Table 12.3. Examples of molecular (Table 12.5).
methods that have been or could be used to detect these Several approaches have been taken to test for the
gene lesions are also listed. Not all of these methods are Leiden mutation. Polymerase chain reaction (PCR)
in common use in molecular diagnostics. Some diseases methods include the use of restriction fragment length
are effectively analyzed by morphological studies or polymorphism (PCR-RFLP) or PCR with sequence-
clinical chemistry. For instance, hemoglobin S is classi- specific primers (SSP-PCR; Figs. 12.7 and 12.8).
cally detected by protein electrophoresis. Final diagnosis Nonamplification molecular methods, such as Invader
requires physiological, morphological, and laboratory technology, have also been developed to test for this
results. gene mutation. Clot-based methods may be used to
directly demonstrate thrombophilia before genetic
testing. Family history may also be considered.
Lysosomal Storage Diseases
Lysosomes are subcellular organelles in which prod-
Prothrombin
ucts of cellular ingestion are degraded by acid hydrolase
enzymes (Fig. 12.6). These enzymes work in an acid Prothrombin is the precursor to thrombin in the coag-
environment. Substrates come from intracellular turn- ulation cascade and is required for the conversion
over (autophagy) or outside the cell through phagocy- of fibrinogen to fibrin. A mutation in the 3′ untrans-
tosis or endocytosis (heterophagy). Lysosomal storage lated region of the gene that codes for prothrombin
disorders result from incompletely digested macromol- or coagulation factor II, F2 (11p11-q12), results in an
ecules due to loss of enzymatic degradation. Storage autosomal-dominant increased risk of thrombosis (see
disorders include defects in proteins required for normal Table 12.5). Laboratories may test for both F2 and F5
lysosomal function, giving rise to physical abnormali- mutations. Both may be present in the same individ-
ties. The organs affected depend on the location and ual, in which case the risk of thrombosis is greater than
site of degradation of the substrate material. Examples with one of the mutations alone. An example of a multi-
of storage diseases are shown in Table 12.4. These dis- plex PCR-RFLP method to simultaneously test for both
orders are screened by gene product testing, that is, mutations was described previously in Chapter 8. In this
measuring the ability of serum enzymes to digest test method, primers that amplify prothrombin and factor V
TABLE 12.3 Single-Gene Disorders and Molecular Methods
Examples of
Type of Type of Molecular
Protein Type of Disease Example Gene (Location) Mutation Methods
Structural Hemoglobinopathies Sickle cell anemia Hemoglobin beta Missense Sequencing,

(11p15.5) PCR-RFLP
Connective tissue Marfan syndrome Fibrillin (15q21.1) Missense Sequencing,

disorders linkage analysis
Cell membrane– Muscular dystrophy Dystrophin, DMD Deletion Southern blot

associated protein (Xp12.2) RFLP,21 multiplex
dysfunction PCR, linkage
analysis
Cell surface Hypercholesteremia Familial Low-density Deletions, point Probe

receptor hypercholesteremia lipoprotein receptor mutations amplification,
proteins (19p13.2) sequencing
Nutritional disorders Vitamin D–resistant Vitamin D receptor Point mutations Southern blot
rickets (12q12-q14) RFLP, sequencing
Cell growth Fibromas Neurofibromatosis type Neurofibromin Missense, Sequencing,

regulators 1 (von Recklinghausen tumor suppressor frameshift, splice linkage analysis
disease) (17q11.2) site mutations
Fibromas Neurofibromatosis type Merlin tumor Nonsense, Linkage analysis

2 (von Recklinghausen suppressor, NF-2 frameshift, splice
disease) (22q12) site mutations
Cancer Li-Fraumeni syndrome p53 tumor- Missense Sequencing, SSCP,

predisposition (LFS)* suppressor gene, mutations DGGE
TP53 (17p13)
Enzymes Metabolic diseases Alkaptonuria Homogentisic acid Missense, cDNA sequencing,

(ochronosis) oxidase (3q21–q23) frameshift, splice SSCP
site mutations
Phenylketonuria Phenylalanine Splice site, missense Ligase chain

hydroxylase, PAH or mutations, reaction,25,26
PKU1 (12q24.1) deletions direct sequencing
Immunodeficiencies Severe combined Adenosine Point mutations Direct sequencing,

immunodeficiency deaminase capillary
(20q13.11) electrophoresis
*A significant proportion of LFS and LFL (Li-Fraumeni–like) kindred do not have demonstrable TP53 mutations.
are designed to destroy or produce HindIII restriction each lane reveal the F2 and F5 normal or mutant geno-
sites in the presence of the F5 or F2 mutation, respec- types for each specimen.4
tively. The sizes of the amplicons and their restriction Thrombin time, prothrombin time, platelet count, and
fragments allow resolution of both simultaneously by complete blood count are phenotypic tests that may be
agarose gel electrophoresis. The fragment patterns in performed in addition or prior to molecular analysis.
Phagocytosis
Food
particles
Phagosome Lysosome
Products of Golgi
digestion apparatus
Undigested
material Autophagy Nucleus
Cellular
material
Recycled
material
FIGURE 12.6 The lysosome is a depository for cell debris. The lysosome contains enzymes that are active in its acid environ-
ment to digest proteins delivered from phagocytosis of foreign bodies, endocytosis, and autophagy of internal cellular components
such as mitochondria.
TABLE 12.4 Storage Diseases TABLE 12.5 Risk of Thrombosis Relative

to Normal (1) Under the Indicated Genetic
Substrate Accumulated Disease (F5, Prothrombin) and Environmental
(OCP) Influences
Sphingolipids Tay–Sachs disease
Glycogen Von Gierke, McArdle, and Status Risk of Thrombosis

Pompe disease
Normal 1
Mucopolysaccharides Hurler, Sheie (MPS I), Hunter
(MPS II), Sanfilippo (MPS III), Oral contraceptive (OCP) use 4
Morquio (MPS IV), Maroteaux–
Lamy (MPS VI), Sly (MPS VII) Prothrombin mutation, 3
heterozygous
Mucolipids Pseudo-Hurler polydystrophy
Prothrombin mutation + OCP 16
Sulfatides Niemann–Pick disease
R506Q heterozygous 5–7
Glucocerebrosides Gaucher disease
R506Q heterozygous + OCP 30–35
R506Q homozygous 80
R506Q homozygous + OCP 100+

Exon 10 Exon 10
F5 gene F5 gene
MnlI site MnlI site

Mutant …A…
Normal …G…
Normal …G…
MnlI site
MnlI site destroyed
Mutant …A…
+/+ +/m m/m
148 bp
+/+ +/m m/m 123 bp
153 bp
116 bp
FIGURE 12.8 In sequence-specific PCR, a primer with thy-
midine as its final 3′ base will yield a product only if the
67 bp
adenine nucleotide is present. The resulting 148 bp PCR
product reflects the presence of the mutation. By designing a
primer slightly shorter than but complementary to the normal
37 bp (G) in the template, a distinct, shorter 123-bp normal product
is amplified. In a heterozygous individual, both products will
FIGURE 12.7 PCR-RFLP for the factor V Leiden mutation. appear.
The R506Q amino acid substitution is caused by a G to A
change in exon 10 of the F5 gene. This DNA mutation destroys
an MnlI restriction enzyme site. An amplicon including the site co-substrate for conversion of homocysteine to methi-
of the mutation, when cut with MnlI, will yield three fragments onine (Fig. 12.9). Two (of more than a dozen) genetic
in normal DNA (+/+) and two products in homozygous mutant
polymorphisms, 677C>T (p.A222V) and 1298 A>C
DNA (m/m). A heterozygous specimen (+/m) will yield a com-
bination of the normal and mutant pattern.
(p.E429A), are associated with deficiencies in folate
metabolism. These variants are detectable by standard
or multiplex PCR with RFLP using restriction enzymes
Automated systems that measure changes in light trans- HinfI and MboII or sequencing. Multiplex qPCR and
mittance during clot formation generating a curve for high-resolution melt-curve methods have also been
mathematical analysis replace some of the manual developed.5,6
methods. To further identify the genetic cause of abnor-
mal coagulation, sequencing of factors IX and XIII is
Hemochromatosis
performed in addition to factor V and II analysis.
Hemochromatosis is an autosomal-recessive condition
that causes overabsorption of iron from food. Iron accu-
Methylenetetrahydrofolate Reductase
mulation subsequently causes pancreas, liver, and skin
Deficiency of the 5,10-methylenetetrahydrofolate reduc- damage; heart disease; and diabetes. Classically, diagno-
tase (MTHFR) gene product is an autosomal-recessive sis is made through measurement of blood iron levels,
disorder that results in increased homocysteine levels transferrin saturation, or liver biopsy.
(hyperhomocysteinemia), causing a predisposition to At the molecular level, hemochromatosis is caused
venous and arterial thrombosis. by dysfunction of the hemochromatosis type I HFE or
MTHFR catalyzes the conversion of 5,10- HLA-H gene product. HFE (6p21.3) codes for a mem-
methylenetetrahydrofolate to 5-methyltetrahydrofolate, a brane-bound protein that binds with β2 microglobulin
Methylene THF
Outside of cell H63D and S65C mutations
MTHFR
5-methylTHF α1 heavy chain

S S α2
Methionine
Methionine synthase
S-adenyl- Homocysteine NH2
NH2
methionine β2 -microglobulin
S
S
S α3
S
Methylation
reactions Cell membrane HOOC C282Y mutation
FIGURE 12.9 MTHFR catalyzes the conversion of

5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a
co-substrate for homocysteine-mediated remethylation to
methionine. Mutations in this gene are associated with throm-
bophilia and methylenetetrahydrofolate reductase deficiency.
Cytosol
HOOC
and transferrin on the membrane of cells in the small
intestine and also on the placenta. The protein directs
iron absorption based on cellular iron loads. In the FIGURE 12.10 The HFE protein is associated with
absence of HFE function, intestinal cells do not sense β2-microglobulin in the cell membrane. The location of the fre-
iron stores, and iron absorption continues into overload. quently occurring mutations is shown.
The most frequently observed mutation in hemo-
chromatosis is C282Y, found in approximately 10% CF is caused by loss of function of the CF transmem-
of the Caucasian population, with a disease frequency brane conductance regulator, the CFTR gene (7q31.2).
of 2 to 3 per 1,000 people. Other mutations most fre- The gene codes for a chloride channel membrane protein
quently detected in the HFE protein are H63D and S65C (Fig. 12.12). The first and most frequently observed
(Fig. 12.10). Clinical symptoms and increased serum mutation in CFTR is a 3-bp deletion that removes a
ferritin and transferrin-iron saturation are indications phenylalanine residue from position 508 of the protein
for mutation testing. The C282Y mutation is detectable (F508del).7 More than 1,900 other mutations and varia-
using PCR-RFLP (Fig. 12.11). tions have been reported in and around the CFTR gene
in diverse populations. In a European survey of over
25,000 patients, the seven most frequent mutations were
Cystic Fibrosis
F508del, G542X, G551D, N1303K, R117H, W1282X,
Cystic fibrosis (CF) is a life-threatening autosomal- and 1717-1G->A. These variants accounted for 75%
recessive disorder that causes severe lung damage and of the alleles, with F508del present in 66.7% of the
nutritional deficiencies. With earlier detection by genetic samples.8 A list of mutations, their locations, and refer-
analysis and improved treatment strategies, people with ences is available through the Human Genome Variation
CF can live more comfortably surviving beyond the Society at http://www.genet.sickkids.on.ca.
fourth decade of life. CF affects the cells that produce Genetic testing for CF is important for diagnosis and
mucus, sweat, saliva, and digestive juices. Normally, genetic counseling because early intervention is most
these secretions are thin, but in CF, a defective gene effective in relieving symptoms of the disease. Molec-
causes the secretions to become thick and sticky. Respi- ular tests have been designed to detect a variety of
ratory failure is the most dangerous consequence of CF. mutations that have been described in CF9 and include
Exon 4
HFE gene
Outside of cell
Carbohydrate
Cell membrane
Rsa1 site
Normal …G…
Rsa1 site Rsa1 site
Mutant …A… Cytoplasm
Nucleotide-
binding
Phosphate
+/+ +/+ m/m +/m +/+ +/+ domain
Regulatory
240 bp F508del domain
140 bp
FIGURE 12.12 The CF transmembrane conductance regula-
tor forms a channel in the cell membrane. The F508del and
110 bp other mutations that affect its function are responsible for the
phenotype of CF.
FIGURE 12.11 Detection of the C282Y mutation by
PCR-RFLP. The G→A mutation in exon 4 of the HFE gene
produces a site for the restriction enzyme, Rsa1. This region is
first amplified using primers flanking exon 4 of the gene NADPH NADP⫹
Bilirubin
(arrows). In a normal specimen, the enzyme will produce two
AH ⫹ O2
fragments, 240 bp and 140 bp. If the mutation is present, the Heme FAD
Heme AOH ⫹ H2O
140-bp normal fragment is cut to a 110-bp and a 30-bp frag- oxygenase FMN e⫺
ment (the 30-bp fragment is not shown). Heterozygous indi-
P-450
viduals will have both the 140-bp and the 110-bp fragments. P-450 P-450 P-450
reductase
RFLP, PCR-RFLP, heteroduplex analysis, temporal
temperature-gradient gel electrophoresis, single-strand
conformation polymorphism (SSCP), SSP-PCR, Cleav-
ase, bead array technology, and direct sequencing. The
American College of Medical Genetics (ACMG) and the
American College of Obstetricians and Gynecologists FIGURE 12.13 NADPH cytochrome P-450 reductase cata-
(ACOG) have recommended a core panel of 23 muta- lyzes the reduction of NADPH and transfers electrons to flavin
adenine dinucleotide (FAD). Electrons flow through FAD and
tions that will identify 49% to 98% of carriers, depend-
flavin mononucleotide (FMN) to the cytochromes. The cyto-
ing on ethnic background. Sequencing panels include chromes then oxidize a variety of substrates (AH). NADPH
these and more rare variants.10 Population differences cytochrome P-450 reductase also works with heme oxygenase
and variable expressivity influence the choice of muta- to convert heme to biliverdin and eventually to bilirubin.
tions to be covered.
enzymes are mono-oxygenases; that is, they participate
Cytochrome P-450 in enzymatic hydroxylation reactions and also transfer
Cytochrome P-450 comprises a group of enzymes local- electrons to oxygen:
ized to the endoplasmic reticulum (Fig. 12.13). These A− H + B− H 2 + O2 → A− OH + B + H 2 O
where A is the substrate and B is the hydrogen donor. these polymorphisms.11,12 CYP-450 polymorphisms may
These enzymes influence steroid, amino acid, and drug also compound interactions of multiple drugs taken
metabolism using NADH or NADPH as hydrogen simultaneously.13,14
donors. Oxygenation of lipophilic drugs renders them There are over 30 reported variations of CYP-450
more easily excreted. enzymes.15 Enzymes are classified according to families
The cytochrome P-450 system is present in high con- and subfamilies. For example, CYP2A6 is cytochrome
centrations in the liver and small intestine where the P-450, subfamily IIA, polypeptide 6. CYP1A2 and the
enzymes metabolize and detoxify compounds taken in enzymes in the CYP2 and CYP3 families are consid-
orally (Fig. 12.14). The P-450 system varies from one ered to be most important in drug metabolism. Some of
person to another. This may in part account for differ- the enzymes reported to inhibit or induce drug metab-
ent effects of drugs on different people. The metabo- olism include CYP1A2, CYP2A6, CYP2B6, CYP2C8,
lism of hormones, caffeine, chemotherapeutic drugs, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1,
antidepressants, and oral contraceptives is affected by CYP3A4, and CYP3A5-7. The genes coding for these
enzymes are located throughout the genome.
Drug
Genetic polymorphisms of cytochrome P-450 genes
are unequally distributed geographically and in different
ethnic populations. Testing for these polymorphisms is
used to predict the response to drugs sensitive to metab-
olism by this enzyme system. In the laboratory, testing
CYP-450 for CYP-450 polymorphisms is performed by allele-spe-
cific PCR for particular polymorphisms. Multiple P-450
genetic variants may be screened by microarray, bead
array, or sequencing.
Metabolite
Conjugation
(Kidney) SINGLE-GENE DISORDERS WITH NONCLASSICAL

PATTERNS OF INHERITANCE
(Urine)
Adduct
(Liver) Mitochondrial mutations, genomic imprinting, and
gonadal mosaicism do not follow Mendelian rules
of inheritance. Mitochondrial mutations are inherited
(Intestine) maternally (Fig. 12.15). Genomic imprinting is responsi-
ble for specific expression of genes in different cells and
tissues. Imprinting is reset at meiosis and fertilization
(Stool) and is different in egg and sperm production.
FIGURE 12.14 The oxidation activities of cytochrome P-450 Gonadal mosaicism is the generation of new muta-
proteins metabolize a variety of structurally diverse chemicals, tions in germline cells. The mutated cells give rise
including therapeutic drugs. to eggs or sperm carrying the mutation, which then
FIGURE 12.15 Mitochondrial mutations are

maternally inherited.
FIGURE 12.16 Gonadal mosaicism arises as a result

of a new mutation. In this example, a dominant disease
phenotype has been inherited from two unaffected
parents. The mutation is present only in the germ cells
of the first-generation parents but is inherited in all
cells of the offspring.
becomes a heritable phenotype. Unusual pedigrees result HV 1 HV 2

(Fig. 12.16). Gonadal mosaicism is expected when (342 bp) (268 bp)
phenotypically normal parents have more than one PH1
affected child (e.g., in osteogenesis imperfecta, an auto-
PH2
somal-dominant phenotype in a child from unaffected PL LHON
14484T>C
parents). MELA
3243A>G Deleted
Advanced Concepts LHON
areas
3460G>A
An example of the nature of imprinting is illus- LHON
trated by a comparison of mules and hinnies. A 11778G>A
mule is the product of a male donkey and a female MERRF
horse. A hinny is the product of a male horse and 8344A>G MERRF
a female donkey. These animals are quite differ- 8393T>G
ent in phenotype, even though they contain essen- FIGURE 12.17 Mutations and deletions throughout the mito-
tially the same genotype. Another illustration of chondrial genome are associated with muscular and neurologi-
the effects of imprinting is seen in animals cloned cal disorders.
by nuclear transfer. Because this process bypasses
the generation of eggs and sperm and fertilization, the most energy-demanding organs, the muscles and
imprinting is not reset, and cloned animals display the nervous system.17 Mutations in several genes in the
unexpected phenotypes, such as larger size or mitochondrial genome have been defined. These muta-
early onset of age-related diseases. tions result in a number of disease syndromes involving
muscular and neurological disorders (Table 12.6).
Mutations in Mitochondrial Genes Advanced Concepts

Mitochondria are cellular organelles responsible for
Disease severity depends on the number of mito-
energy production. Mitochondria contain their own
chondria affected. Mitochondria are actively
genome, a circular DNA molecule 16,569 bp in length
undergoing turnover in the cell, including fission
(Fig. 12.17). The mitochondrial genome contains
and fusion, which are part of the mitochondrial
37 genes, including a 12S and 16S rRNA, 22 tRNAs,
network quality control. Alterations in mitochon-
and 13 genes required for oxidative phosphorylation. In
drial dynamics can cause neuropathies or optic
addition, the mtDNA contains a 1000-nt control region
atrophy.18
that encompasses transcription and replication regula-
tory elements. A database of mitochondrial genes and
mutations is available at http://www.MITOMAP.org.16 Mitochondrial mutations are easily detected by a variety
Mutations in mitochondrial genes affect energy proof molecular methods. Southern blot is used for detect-
duction and are therefore manifested as diseases in ing large deletions (Fig. 12.18). Point mutations are
TABLE 12.6 Diseases Resulting From Mutations in Mitochondrial Genes
Disorder Gene Affected Molecular Methodology
Kearns–Sayre syndrome 2–7 kb deletions Southern blot, PCR, PCR-RFLP
Pearson syndrome Deletions Southern blot analysis of

leukocytes, PCR-RFLP
Pigmentary retinopathy, chronic progressive external tRNA (tyr) deletion, deletions PCR-RFLP, Southern blot analysis
ophthalmoplegia of muscle biopsy
Leber hereditary optic neuropathy Cyt6 and URF* point mutations PCR-RFLP
Mitochondrial myopathy, encephalopathy, lactic tRNA (leu) point mutations PCR-RFLP, sequencing
acidosis, and strokelike episodes
Myoclonic epilepsy with ragged red fibers tRNA (lys) point mutations PCR-RFLP
Deafness
Neuropathy, ataxia, retinitis pigmentosa (NARP) ATPase VI point mutation PCR-RFLP
Subacute necrotizing encephalomyelopathy with ATPase VI, NADH:ubiquinone PCR-RFLP

neurogenic muscle weakness, ataxia, retinitis oxidoreductase subunit mutations
pigmentosa (Leigh syndrome with NARP)
Mitochondrial DNA depletion syndrome Thymidine kinase gene mutations PCR, sequencing
*Unknown reading frame
analyzed by PCR-RFLP (Fig. 12.19). Interpretation of with 3-bp repeating units) occur in coding and noncod-
mutation analysis has long been complicated, however, ing sequences of genes. The most well-known examples
by the extent of heteroplasmy (mutated mitochondria of triplet-repeat expansion diseases are fragile X syn-
and normal mitochondria in the same cell) and the nature drome and Huntington disease.
of the mutation.19 A range of phenotypes may be present,
even in the same family. Advanced Concepts
Genes that control mitochondrial functions are also
found on the nuclear genome (Table 12.7). Unlike mito- In addition to family history, clinical tests includ-
chondrial mutations that display maternal inheritance, ing electroencephalography, neuroimaging, cardiac
these disorders have autosomal patterns of inheritance. electrocardiography, echocardiography, magnetic
Although the causative gene mutation is located on a resonance spectroscopy, and exercise testing are
nuclear gene, analysis of mitochondria may still show important for the accurate diagnosis of mito-
deletions or other mutations caused by the loss of the chondrial disorders. High blood or cerebrospinal
nuclear gene function. fluid lactate concentrations, as well as high blood
glucose levels, are observed in patients with some
mitochondrial diseases. More direct tests, such
Nucleotide-Repeat Expansion Disorders
as histological examination of muscle biopsies
Nucleotide repeats include short tandem repeats (STRs) and respiratory chain complex studies on skeletal
with 1 to 10-bp repeating units. During DNA replica- muscle and skin fibroblasts, are more specific for
tion and meiosis, these STRs can expand (or contract) mitochondrial dysfunction.
in length. Triplet-repeat mutations (expansions of STR
Fragile X Syndrome
Fragile X syndrome is associated with a triplet-repeat
(CGG) expansion in the noncoding region 5′ to the
fragile X mental retardation gene, FMR-1 (Fig. 12.20).
The expansion becomes so large in full fragile X syn-
drome (more than 2,000 CGG repeats) that the region
is microscopically visible (Fig. 12.21). The CGG repeat
expansion 5′ to the FMR-1 gene also results in meth-
ylation of the region and transcriptional shutdown of
Spec. 1 2 3
Mspl U C U C U C
551 bp
FIGURE 12.18 A mitochondrial deletion as revealed by 345 bp
Southern blot. DNA was cut with PvuII, a restriction enzyme 206 bp
that cuts once in the mitochondrial genome. The membrane
was probed for mitochondrial sequences. Normal mitochondria
(N) yield one band at 16.6 kb when cut with PvuII (C). Super-
coiled, nicked, and a few linearized mitochondrial DNA circles
can be seen in the uncut DNA (U). DNA from a patient with
Kearns–Sayres syndrome (P) yields two mitochondrial popula-
tions, one of which has about 5 kb of the mitochondrial FIGURE 12.19 Detection of the NARP mitochondrial point
genome–deleted sequences. Because both normal and mutant mutation (ATPase VI 8993T>C or T>G) by PCR-RFLP. The
mitochondria are present, this is a state of heteroplasmy. (Photo PCR product was digested with the enzyme MspI (C) or undi-
courtesy of Dr. Elizabeth Berry-Kravis, Rush University Medical gested (U). If the mutation is present, the enzyme will cut the
Center.) PCR product into two pieces, as seen is specimen 3. (Photo
courtesy of Dr. Elizabeth Berry-Kravis, Rush University Medical
Center.)
TABLE 12.7 Some Disorders Caused by Nuclear Gene Mutations
Disorder Gene Affected (Location) Molecular Methodology
Mitochondrial DNA depletion syndrome Succinate-CoA ligase, ADP-forming, beta subunit, Southern blot
SUCLA 2 (13q12.2-q13)
Mitochondrial neurogastrointestinal Platelet-derived endothelial cell growth factor, Sequencing

encephalomyopathy ECGF (22q13-qter)
Progressive external ophthalmoplegia Chromosome 10 open reading frame 2, C10ORF2 Southern blot, SSCP, sequencing
(10q24); polymerase, DNA, gamma, POLG
(15q25); solute carrier family 25 (mitochondrial

carrier), member 4, SLC25A4/ANT1 (4q25)
Normal
5′ CGG(CGG)5–55 FMR-1 3′
Amplification
Premutation (carrier)
5′ CGGCGGCGG(CGG)56–200 FMR-1 3′
Amplification and methylation

Full mutation (affected)
5′ CGGCGGCGGCGGCGGCGG(CGG)200–2000+ FMR-1 3′
Unaffected carrier
FIGURE 12.20 Triplet-repeat (CGG) expansions in Learning disabled
sequences 5′ to the FMR-1 gene are observed in fragile X car-
FXS
riers (up to 200 repeats) and fully affected individuals (more
than 200 repeats). Normally there are fewer than 60 repeats.
Expansion results from amplification of the triplet sequences
FIGURE 12.22 The symptoms of fragile X syndrome (FXS)
become more severe with each generation. The fragile X chro-
during meiotic recombination events. The very large repeats
mosome cannot be transmitted from fathers to sons but can be
(more than 200 repeats) are methylated on the C residues. This
transmitted from grandfathers to grandsons through their
methylation turns off FMR-1 transcription.
daughters.
primary ovarian insufficiency (FXPOI), with amenor-
rhea, menopausal follicle-stimulating hormone (FSH)
levels, and possible estrogen deficiency.20 Another con-
dition associated with PM status is fragile X tremor and
ataxia syndrome (FXTAS), with declining overall cogni-
tive abilities with age.21
In addition to the fragile X chromosome observed
by karyotyping, the state of the repeat expansion is also
analyzed using PCR and by Southern blot (Fig. 12.23).
Premutations in fragile X carriers are easily detected
by PCR, with full mutations detectable by triple-repeat-
primed PCR. AGG interruptions can be detected with
Fragile X chromosome AGG-triplet-repeat primers.22 Southern blot can reveal
(in metaphase) cases of mosaicism where both premutations and full
FIGURE 12.21 The fragile X chromosome is characterized fragile X chromosomes are present in separate cell pop-
by a threadlike process just at the telomere of the long arm ulations from the same patient.
(arrow). This is the site of disorganization of chromatin struc- Capillary electrophoresis is an increasingly popular
ture by the GC-rich repeat expansions. option for identifying expanded FMR alleles, replacing
the gel procedures (Fig. 12.24). Peak patterns indicate
FMR-1. CGG repeats can be interrupted by AGG, which the presence of normal, premutation, and full fragile
dampens the methylation and silencing of FMR-1. X mutation. The presence of AGG interrupters of the
Symptoms of fragile X syndrome include learn- CGG repeats shows as gaps in the series of peaks. AGG
ing disorders and mental retardation (IQ ~20), long promotes stability or slower expansion of the repeat
face, large ears, and macroorchidism (large genitalia). region. The capillary electrophoresis method is faster and
Symptoms are more apparent at puberty. Symptoms technically less demanding than Southern blot; however,
increase in severity with each generation in a fragile the latter procedure may still be required to identify the
X family (Fig. 12.22). Approximately 20% of women presence of deletions within the FMR-1 gene or the 5′
with FMR-1 premutation (PM) will develop fragile X repeats in a percentage of the cells (mosaicism).
PCR Southern blot
50–90
20–40
Inactive X
Premutations Full mutations
FIGURE 12.23 Detection of premutations by PCR (left) and full fragile X mutations by Southern blot (right). Primers (one of
which is labeled with 32P) flanking the repeat region are used to generate labeled PCR products. Premutations appear as large
amplicons in the 50- to 90-repeat range on the autoradiogram at left. Normal samples fall in the 20- to 40-repeat range. Full fragile
X repeats are too large and GC rich to detect by standard PCR. Southern blots reveal full mutations in three of the samples shown.
The inactive (methylated) X chromosome in four female patients is detected by cutting the DNA with a methylation-specific
restriction enzyme. (Photos courtesy of Dr. Elizabeth Berry-Kravis, Rush University Medical Center.)
FU
A 150 200 250 300 350 400
FU
B 150 200 250 300 350 400
FU
C 150 200 250 300 350 400
FIGURE 12.24 Fragile X premutations and full mutations appear as altered peak patterns in an electropherogram. PCR products
of the FMR-1 CGG repeat region in the normal X chromosome (A) are detected as peaks of less than 250 bases (A), whereas the
premutation (B) and full mutation (C) are visible as regular peaks extending up to 400 bases.
Huntingtin
80–170 bp
FIGURE 12.25 The huntingtin repeat expansion P
occurs within the coding region of the huntingtin ⬎40 repeats
gene. The expansion is detected directly by PCR
using primers flanking the expanded region (top). A
32
P-labeled primer is used, and the bands are detected
by autoradiography of the polyacrylamide gel
(bottom). In this example, PCR products from the
10–29 repeats
patient (P) fall within the normal range with the neg-
ative control (–). The positive control (+) displays
the band sizes expected in Huntington disease.
(Photos courtesy of Dr. Elizabeth Berry-Kravis, Rush Uni-
versity Medical Center.)
Huntington Disease In contrast to fragile X, where the repeat expansion

takes place 5′ to the coding sequences, the Huntington
Huntington disease, first described by George Hunting-
expansion occurs within the coding region of the gene.
ton in 1872, is associated with expansion within the
The triplet expansion inserts multiple glutamine residues
huntingtin structural gene (4p16.3). In this repeat expan-
in the 5′ end of the huntingtin protein. This causes the
sion, the sequence CAG expands from 9 to 37 repeats to
protein to aggregate in plaques, especially in nervous
38 to 86 in the huntingtin gene. The clinical symp-
tissue, causing the neurological symptoms seen in this
toms of Huntington disease include impaired judgment,
disease. The expansion does not reach the size of the
slurred speech, difficulty in swallowing, abnormal body
fragile X expansion and is detectable by standard PCR
movements (chorea), personality changes, depression,
methods and capillary electrophoresis (Fig. 12.25).
mood swings, unsteady gait, and an intoxicated appear-
ance. With onset in the 30s or 40s, these symptoms do
Idiopathic Congenital Central
not become obvious until the fourth or fifth decade of
Hypoventilation Syndrome
life, usually after family planning. The child of a person
with Huntington disease has a 50% chance of inheriting Idiopathic congenital central hypoventilation syndrome
the disorder. Genetic counseling, therefore, is important (CCHS) is a rare pediatric disorder characterized by
for younger persons with family histories of this disease, inadequate breathing while asleep. More-affected chil-
especially with regard to having children. dren may also experience hypoventilation while awake.
CCHS occurs in association with an intestinal disorder
called Hirschsprung disease and symptoms of diffuse
autonomic nervous system dysregulation/dysfunction.
Advanced Concepts A number of gene mutations have been observed in
CCHS, including a polyalanine expansion of the paired-
The FMR protein (FMRP) binds RNA and is asso- like homeobox (PHOX2b) gene (4p12)13. The PHOX2b
ciated with polysomes. FMRP regulates translation protein is a transcription factor containing a domain
of its bound mRNAs through alternative mRNA (homeobox) similar to other proteins that bind DNA.
splicing, mRNA stability, and mRNA trafficking In CCHS, a triplet-repeat expansion occurs inside of
from the nucleus to the cytoplasm. FMRP may be the PHOX2b gene, resulting in the insertion of multi-
associated with the miRNA pathway as well, pre- ple alanine residues into the protein. The severity of the
venting helicase-mediated miRNA suppression.23,24 disease increases with increasing numbers of repeats.
The expansion is detected by PCR (Fig. 12.26).25
PHOX2b exon 3
PAGE
(Normal)
(Normal)
Agarose
(rapid test)
FIGURE 12.26 The triplet-repeat expansion of PHOX2b includes triplets that code for alanine (top). The expansion is detected
by PCR with a 32P-labeled primer and polyacrylamide gel electrophoresis (center) or by standard PCR and agarose gel electropho-
resis (bottom). Normal specimens yield a single PCR product. CCHS specimens yield another larger product in addition to the
normal product. The standard PCR test can rapidly show the presence of the expansion, and the PAGE test allows determination
of the exact number of alanine codons that are present in the expansion. (Photos courtesy of Dr. Elizabeth Berry-Kravis, Rush University
Medical Center.)
CCHS is usually apparent at birth. In some children, The difference is exhibited in genetic disorders in which
late onset of the disease occurs at 2 to 4 years of age. An one or the other allele of a gene is lost.
estimated 62% to 98% of patients with CCHS have the A uniparental disomy/deletion demonstrates the
PHOX2b gene mutation.26 nature of imprinting on chromosome 15. A deletion in
the paternal chromosome 15, del(15)(q11q13), causes
Prader–Willi syndrome. Symptoms of this disorder
Other Nucleotide Expansion Disorders
include mental disability, short stature, obesity, and
Fragile X, Huntington disease, and CCHS are three of hypogonadism. Loss of the same region from the mater-
a group of diseases caused by trinucleotide-repeat dis- nal chromosome 15 results in Angelman syndrome, a
orders. This category of diseases is subclassified into disorder with very different symptoms, including ataxia,
polyglutamine expansion disorders, which includes seizures, and inappropriate laughter. Both syndromes
Huntington disease, and non–polyglutamine expansion can occur in four ways: a deletion on the paternal or
disorders. Larger repeat units may also be involved in maternal chromosome 15, a mutation on the paternal or
expansion mutations. Expansion of a hexanucleotide maternal chromosome 15, a translocation with loss of
repeat unit is found in cases of amyotrophic lateral scle- the critical region from one chromosome, and maternal
rosis (ALS).27 Examples of repeat expansion diseases or paternal uniparental disomy in which both chromo-
are listed in Table 12.8. somes 15 are inherited from the mother and none from
the father or vice versa.
Cytogenetic methods are used to test for these genetic
Genomic Imprinting
lesions. Translocations and some deletions are detect-
Genomic imprinting is transcriptional silencing through able by standard karyotyping. High-resolution karyotyp-
histone or DNA modification. Imprinting occurs during ing can detect smaller deletions; however, other cases
egg and sperm production and is different in DNA are not detectable microscopically. FISH with labeled
brought in by the egg or the sperm upon fertilization. probes to the deleted region can detect over 99% of
TABLE 12.8 Examples of Nonpolyglutamine Expansion Disorders
Expansion, (Normal)—
Disorder Gene Repeat (Symptomatic)*
Fragile XE Fragile X mental retardation 2 (Xq28) GCC (6–35) – (over 200)
Friedreich ataxia Frataxin, FRDA or X25 (9q13) GAA (7–34) – (over 100)
Myotonic dystrophy Dystrophia myotonica protein kinase (9q13.2–13.3) CTG (5–37) – (over 50)
Spinocerebellar ataxia type 8 Spinocerebellar ataxia type 8 (13q21) CTG (16–37) – (110–250)
Spinocerebellar ataxia type 12† Spinocerebellar ataxia type 12 (5q31–33) CAG (7–28) – (66–78)
Amyotrophic lateral sclerosis, C9 open reading frame 72 (9p21.1) GGGGCC (2–20) – (over 100)
frontotemporal disorder
*The phenotypic effects of intermediate numbers of repeats is not known.

†Although CAG codes for glutamine, this expansion occurs outside of the coding region of this gene and is not translated.
cases. PCR of RFLP or STR analysis has also been used combinations of genetic (and somatic) variants. Further
to demonstrate uniparental disomy. Because imprinting annotation of demographics, such as ethnicity or gender,
(DNA methylation) is different on maternal and paternal and lifestyles, such as smoking or diet, will further add
chromosomes, methylation-specific PCR and Southern to the prognostic and diagnostic value of gene mutation
blot using methylation-specific restriction enzymes can analysis in these cases.
aid in the diagnosis of these disorders. Assays developed
for the detection of copy-number variants, including
FISH, array-based comparative genomic hybridization LIMITATIONS OF MOLECULAR TESTING
(aCGH), and next-generation sequencing (NGS), have
been used for the detection of genome-wide uniparental Although molecular testing for inherited diseases is
disomy (UPD) associated with constitutional and neo- extremely useful for early diagnosis and genetic counsel-
plastic disorders.27 ing, there are circumstances in which genetic testing may
not be the optimal methodology. To date, most therapeutic
targets are phenotypic so that treatment is better directed
Multifactorial Inheritance
to the phenotype. In genes with variable expressivity,
Most disorders (and normal conditions) are controlled finding a gene mutation may not predict the severity of
by multiple genetic and environmental factors. Multifac- the phenotype. For instance, clotting time and transferrin
torial inheritance is displayed as a continuous variation saturation are better guides for anticoagulant treatment
in populations, with a normal distribution, rather than than the demonstration of the causative gene mutations.
as a specific inheritance pattern. Nutritional or chemi- Molecular testing may discover genetic lesions in the
cal exposures alter this distribution. The range may be absence of symptoms. This raises a possible problem as
discontinuous, with a threshold of manifestation. The to whether treatment is indicated. This is increasingly
phenotypic expression is conditioned by the number of possible with the use of large array or sequencing panels
controlling genes inherited. The chance of a first-degree targeting hundreds of genes. Several genetic lesions may
relative having a similar phenotype is 2% to 7%. be present or polymorphic states of other normal genes
High-resolution array methods and next-genera- may influence the disease state. Research methods and
tion sequencing have further defined the genetic com- clinical trials using array technology and sequencing
ponents of multifactorial illnesses. Large databases methods designed to scan at the genomic level may con-
such as ClinVar and dbSNP aid in the interpretation of tribute to better diagnosis of complex diseases.
Case Study 12.1 Case Study 12.2

A young mother was worried about her son. A 14-year-old girl with muscle weakness and
Having observed others, she was very aware of vision difficulties (retinopathy) was referred for
how her baby was expected to grow and acquire clinical tests. A muscle biopsy was performed,
basic skills. As the child grew, however, he showed and aberrant mitochondria were observed in thin
signs of slow development. His protruding ears sections. Histochemical analysis of the muscle
and long face were becoming more noticeable as tissue revealed cytochrome oxidase deficiency in
well. The pediatrician recommended chromosomal the muscle cells. A skeletal muscle biopsy speci-
analysis for the mother and child. A constriction men was sent to the molecular genetics laboratory
at the end of the X chromosome was found in for analysis of mitochondrial DNA. Southern blot
the son’s karyotype. The mother ’s karyotype was analysis of PvuII cut mitochondrial DNA exhib-
normal 46,XX. A Southern blot analysis for fragile ited a band at 16,000 bp in addition to the normal
X was performed on a blood specimen from the mitochondrial band at 16,500 bp, as follows:
mother but showed no obvious abnormality. PCR
analysis produced the following results:
Southern blot of mitochondrial DNA uncut (U) and cut with

PvuII (C). Lanes 1 and 2, normal control; lanes 3 and 4, patient.
Arrow points to a 16,000-kb band.
PCR analysis of the FMR promoter region showing two normal
patterns (lanes 1 and 2) and the mother ’s pattern. QUESTIONS:
QUESTIONS: 1. What is the 16,000 bp product? What is the
11,500 bp product?
1. What do the PCR results in lane 3 indicate?
2. What condition is associated with a 5 kb mito-
2. Why were there no abnormalities detected by
chondrial deletion?
Southern blot?
3. Is this a case of homoplasmy or heteroplasmy?
3. How would this result be depicted using triplet-
primed PCR and capillary electrophoresis?

A 40-year-old man was experiencing increas- A 30-year-old woman was brought to the emer-
ing joint pain, fatigue, and loss of appetite. He gency room with a painfully swollen left leg. She
became alarmed when he suffered heart problems informed the nurses that she was taking contra-
and consulted his physician. Routine blood tests ceptives. Deep vein thrombosis was suspected,
revealed high serum iron (900 μg/dL) and 80% and compression ultrasound was ordered to look
transferrin saturation. Total iron-binding capacity for pulmonary embolism. A clotting test for APC
was low (100 μg/dL). The man denied any exten- resistance resulted in a 1.5 ratio of clotting time
sive alcohol intake. A blood specimen was sent to with and without APC. An enzyme-linked immu-
the molecular genetics laboratory for analysis. The nosorbent assay (ELISA) test for D-dimer was
following results were produced: positive. The patient was immediately treated with
heparin, and blood samples were taken. A blood
sample was sent to the molecular genetics labo-
ratory to test for the factor V Leiden 1691 G→A
mutation and prothrombin 20210 G→A mutations.
The following results were produced:
Prothrombin
PCR-RFLP analysis of exon 4 of the HFE gene. The PCR

product contains 1 recognition site for the restriction enzyme, Factor V
RsaI. An additional RsaI site is created by the C282Y mutation,
the most common inherited mutation in hemochromatosis. This Agarose gel electrophoresis showing the band pattern for pro-
extra site results in fragments of 240 bp, 110 bp (arrow), and thrombin 20210/factor V Leiden mutation detection by multi-
← 30 bp (not shown) instead of the 240-bp and 140-bp normal plex SSP-PCR-RFLP. Specimens were cut with HindIII. Lane
fragments. Lane 1, molecular-weight markers; lane 2, normal 1, molecular-weight marker; lane 2, normal control (prothrom-
control; lane 3, homozygous C282Y; lane 4, heterozygous bin, 407 bp + 99 bp, factor V, 241 bp); lane 3, heterozygous
C282Y; lane 5, patient specimen; lane 6, reagent blank. control, prothrombin (407, 384, 99, 23 bp)/factor V Leiden
(241, 209, 323 bp); lane 4, homozygous factor V Leiden,
QUESTIONS: normal prothrombin; lane 5, homozygous prothrombin 20210
1. Does this patient have the C282Y mutation? mutation/normal factor V; lane 6, patient specimen; lanes 7 and
2. Is this mutation heterozygous or homozygous? 8, normal specimens.
3. Based on these results, what is the likely expla- QUESTIONS:
nation for the patient's symptoms? 1. Does this patient have a clotting disorder?
2. Is there a factor V mutation? Is there a pro-
thrombin mutation?
3. If present, is either mutation homozygous or
heterozygous?
the mutation site. What would you expect from a

STUDY QUESTIONS PCR-RFLP analysis for this mutation in a patient
with MELAS?
1. Which of the following is not a triplet-repeat a. A single PCR product resistant to digestion with
expansion disorder? ApaI
a. Fragile X syndrome b. A single PCR product that cuts into two
b. Huntington disease fragments upon digestion with ApaI
c. Factor V Leiden c. A single PCR product only if the mutation is
d. Congenital central hypoventilation syndrome present
d. Two PCR products
2. A gene was mapped to region 3, band 1, subband
1, of the long arm of chromosome 2. How would 8. A father affected with a single-gene disorder and
you express this location from an idiogram? an unaffected mother have four children (three
boys and a girl), two of whom (one boy and the
3. Which of the following can be detected by PCR? girl) are affected. Draw the pedigree diagram for
this family.
a. Large mitochondrial deletions
b. Full fragile X disorder D16S539, an STR, was analyzed in the family. The
c. Mitochondrial point mutations result showed that the father had the 6,8 alleles,
and the mother had the 5,7 alleles. The affected
4. A patient was tested for Huntington disease. PCR children had the 5,6 and 6,7 alleles, and the
followed by PAGE revealed 25 CAG units. How unaffected children had the 5,8 and 7,8 alleles.
should the results be interpreted? a. If D16S539 is located on chromosome 16,
a. This patient has Huntington disease. where is the gene for this disorder likely to be
b. This patient has a 1/25 chance of contracting located?
Huntington disease. b. To which allele of D16S539 is the gene linked?
c. This patient is normal at the Huntington locus. How might one perform DNA analysis for the
d. The test is inconclusive. presence of the disorder?
5. Which of the following methods can detect the a. Analyze D16S539 for the 6 allele by PCR.
factor V Leiden mutation? b. Sequence the entire region of the chromosome
where D16S539 was located.
a. PCR-RFLP c. Test as many STRs as possible by PCR.
b. SSP-PCR d. Use a variant-specific test to detect the
c. Invader technology unknown gene mutation.
d. All of the above
9. Exon 4 of the HFE gene from a patient suspected
6. The most frequently occurring mutation in the of having hereditary hemochromatosis was
HFE gene results in the replacement of cysteine amplified by PCR. The G to A mutation, frequently
(C) with tyrosine (Y) at position 282. How is found in hemochromatosis, creates an Rsa1 site in
this expressed according to the recommended exon 4. When the PCR products are digested with
nomenclature? Rsa1, which of the following results would you
expect to see if the patient has the mutation?
7. MELAS is a disease condition that results from
an A to G mutation at position 3243 of the a. None of the PCR products will be cut by Rsa1.
mitochondrial genome. This change creates a single b. There will be no PCR product amplified from
ApaI restriction site in a PCR product, including the patient DNA.
c. The patient’s PCR product will yield extra 6. Liew M, Wittwer C, Voelkerding KV. Nucleotide extension
bands upon Rsa1 digestion. genotyping by high-resolution melting. Journal of Molecular
Diagnostics 2010;12:731–738.
d. The normal control PCR products will yield 7. Kerem B, Buchanan JA, Durie P, Corey ML, Levison H, Rommens
extra Rsa1 bands compared with the patient JM, Buchwald M, Tsui L-C. DNA marker haplotype association
sample. with pancreatic sufficiency in cystic fibrosis. American Journal of
Human Genetics 1989;44:827–834.
10. Most people with the C282Y or H63D HFE gene 8. De Boecka K, Zolin H, Cuppens H, Olesen HV, Viviani L. The
relative frequency of CFTR mutation classes in European patients
mutations develop hemochromatosis symptoms. with cystic fibrosis. Journal of Cystic Fibrosis 2014;13:403–409.
This is a result of 9. McGinniss M, Chen C, Redman JB, Buller A, Quan F, Peng M,
Giusti R, Hantash FM, Huang D, Sun W, Strom CM. Extensive
a. iron loss. sequencing of the CFTR gene: lessons learned from the first
b. excessive drinking. 157 patient samples. Human Genetics 2005;118:331–338.
c. high penetrance. 10. Elliott A, Radecki J, Moghis B, Li X, Kammesheidt A. Rapid
d. healthy lifestyle. detection of the ACMG/ACOG-recommended 23 CFTR disease-
e. glycogen accumulation. causing mutations using ion torrent semiconductor sequencing.
Journal of Biomolecular Technology 2012;23:24–30.
11. Haraya K, Kato M, Chiba K, Sugiyama Y. Prediction of inter-
11. The majority of disease-associated mutations in the individual variability on the pharmacokinetics of CYP1A2 sub-
human population are strates in non-smoking healthy volunteers. Drug Metabolism and
Pharmacokinetics 2016;31:276–284.
a. autosomal dominant. 12. Parra-Guillen Z, Berger PB, Haschke M, Donzelli M, Winogra-
b. autosomal recessive. dova D, Pfister B, Früh M, Gillessen S, Krähenbühl S, Kloft C,
c. X-linked. Joerger M. Role of Cytochrome P450 3A4 and 1A2 phenotyping
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Erlotinib treatment. Basic & Clinical Pharmacology & Toxicology
2017;121:309–315.
12. Bead array technology is most appropriate for 13. McDonald M, Au NT, Rettie AE. P450-based drug-drug interac-
which of the following? tions of amiodarone and its metabolites: diversity of inhibitory
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c. STR linkage analysis tives containing ethinyl estradiol and gestodene markedly increase
d. Restriction fragment length polymorphisms plasma concentrations and effects of tizanidine by inhibiting
cytochrome P450 1A2. Clinical Pharmacology and Therapeutics
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2000;2:153–157. drial dynamics in mitochondrial diseases. Diseases 2017;5:ii.
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real-time multiplex polymerase chain reaction detection of meth- falls in the detection of pathogenic mitochondrial DNA mutations.
ylenetetrahydrofolate reductase (MTHFR) 677c>t and 1298a>c Journal of Molecular Diagnostics 2003;5:197–208.
polymorphisms using nearest neighbor model-based probe design. 20. Man L, Lekovich J, Rosenwaks Z, Gerhardt J. Fragile
Journal of Molecular Diagnostics 2007;9:345–350. X-associated diminished ovarian reserve and primary ovarian
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22. Hayward B, Usdin K Improved assays for AGG interruptions in early autonomic nervous system embryologic development and
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Chapter 13
Molecular Oncology
Outline Anaplastic Lymphoma Receptor Tyrosine Kinase (ALK)

Proto-Oncogene, 2p23.1
CLASSIFICATION OF NEOPLASMS V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene
MOLECULAR BASIS OF CANCER Homolog, KIT, c-KIT (4q12)
ANALYTICAL TARGETS OF MOLECULAR TESTING Other Molecular Abnormalities
GENE AND CHROMOSOMAL MUTATIONS IN SOLID TUMORS Microsatellite Instability
Human Epidermal Growth Factor Receptor 2, HER2/neu/ Loss of Heterozygosity
erb-b2 1 (17q21.1) Liquid Biopsy
Epidermal Growth Factor Receptor, EGFR (7p12) MOLECULAR ANALYSIS OF LEUKEMIA AND LYMPHOMA
Kirsten Rat Sarcoma Viral Oncogene Homolog, K-ras (12p12); Gene Rearrangements
Neuroblastoma ras, N-ras (1p13); and Harvey Rat Sarcoma V(D)J Recombination
Viral Oncogene Homolog, H-ras (11p15) Immunoglobulin Heavy-Chain Gene Rearrangement in
Ewing Sarcoma, EWS (22q12) B Cells
Synovial Sarcoma Translocation, Chromosome 18—Synovial Immunoglobulin Light-Chain Gene Rearrangement in
Sarcoma Breakpoint 1 and 2, SYT-SSX1, SYT-SSX2 t(X;18) B Cells
(p11.2;q11.2) T-Cell Receptor Gene Rearrangement
Paired Box-Forkhead in Rhabdomyosarcoma, PAX3-FKHR, Detection of Clonality
PAX7-FKHR, t(1;13), t(2;13) Molecular Analysis of Immunoglobulin Heavy-Chain Gene
Tumor Protein 53, TP53 (17p13) Clonality
Ataxia Telangiectasia Mutated Gene, ATM (11q22) Immunoglobulin Light-Chain Gene Rearrangements
Breast Cancer 1 Gene, BRCA1 (17q21), and Breast Cancer 2 T-Cell Receptor Gene Rearrangements
Gene, BRCA2 (13q12) Banding Patterns
Von Hippel–Lindau Gene, VHL (3p26) Mutations in Hematological Malignancies
V-myc Avian Myelocytomatosis Viral-Related Oncogene, Lymphoid Malignancies
Neuroblastoma-Derived, MYCN or n-myc (2p24) Myeloid Malignancies
V-Ros Avian UR2 Sarcoma Virus Oncogene Homolog 1 (ROS1) Mutation Spectra
Proto-Oncogene (6q22.1) and Rearranged During
Transfection (RET) Proto-Oncogene (10q11)
369
of the primary tumor predicts the likelihood of metas-

Objectives tasis. Both tumor and normal cell factors are involved.
The presence of metastasis increases the difficulty of
13.1 Present basic cancer biology and terminology.
treatment. Clinical analysis may be performed to detect
13.2 Identify checkpoints in the cell division cycle that
the presence of relocated or circulating metastasized
are critical for regulated cell proliferation.
cells to aid in treatment strategy.
13.3 List molecular targets that are useful for
With regard to hematological malignancies, tumors
diagnosing and monitoring solid tumors.
arising from white blood cells are referred to as leuke-
13.4 Explain how microsatellite instability is detected.
mias and lymphomas. Leukemia is a neoplastic disease
13.5 Describe loss of heterozygosity and its detection.
of blood-forming tissue in which large numbers of white
13.6 Contrast cell-specific and tumor-specific molecular
blood cells populate the bone marrow and peripheral
targets.
blood. Lymphoma is a neoplasm of lymphocytes that
13.7 Show how clonality is detected using antibody
forms discrete tissue masses. The difference between
and T-cell receptor gene rearrangements.
these diseases is not clear because lymphocytic leuke-
13.8 Describe translocations associated with
mias and lymphomas can display bone marrow and blood
hematological malignancies that can be used for
symptoms similar to those of leukemias. Furthermore,
molecular testing.
chronic lymphomas can progress to leukemia. Con-
13.9 Interpret data obtained from the molecular
versely, leukemias can display lymphomatous masses
analysis of patients’ cells, and determine if a tumor
without overpopulation of cells in the bone marrow.
population is present.
Within lymphomas, Hodgkin (or Hodgkin’s) disease
is histologically and clinically different from all
other types of lymphoma, termed non-Hodgkin (non-
Oncology is the study of tumors. A tumor, or neoplasm, Hodgkin’s) lymphoma (NHL). Plasma cell neoplasms,
is a growth of tissue that exceeds that of normal tissue which arise from terminally differentiated B cells, are
and is not coordinated with it. Tumors are either benign also classified in a separate category. Some of the phys-
(not recurrent) or malignant (invasive and tending to iological symptoms of plasma cell tumors are related
recur at multiple sites). Cancer is a term that includes to the secretion of immunoglobulin fragments by these
all malignant tumors. Molecular oncology is the study tumors.
of cancer at the molecular level, using techniques that Leukemias arising from undifferentiated cells in the
allow the direct detection of genetic alterations, down to bone marrow are classified as myeloid diseases, such as
single-base-pair changes. acute and chronic myeloid leukemia. Myelodysplastic
syndrome is a dysregulation of cells in a variety of dif-
ferentiation states.
CLASSIFICATION OF NEOPLASMS
Cancer is generally divided into two broad groups, solid Advanced Concepts
tumors and hematological malignancies. Solid tumors
are designated according to the tissue of origin as carci- As imaging technology advanced, several efforts
nomas (epithelial) or sarcomas (bone, cartilage, muscle, were made in the classification of NHL. The ear-
blood vessels, fat). Teratocarcinomas consist of mul- liest was the Rappaport classification in 1966,
tiple cell types. Benign tumors are named by adding developed at the Armed Forces Institute of Pathol-
the suffix -oma to the tissue of origin. For example, an ogy. The Keil classification, used in Europe, and
adenoma is a benign glandular growth. An adenocarci- the Lukes and Collins classification, used in the
noma is malignant. United States, were proposed in 1974. In 1982
Metastasis is the movement of dislodged tumor cells an international group of hematopathologists
from the original (primary) site to other locations. Only proposed the Working Formulation for Clinical
malignant tumors are metastatic. No one characteristic
Chapter 13 • Molecular Oncology 371
receptors transduce signals through the cell membrane

Usage for classification of NHL.1 The Working into the cytoplasm through a series of protein modifi-
Formulation was revised in 1994 by the Interna- cations that ultimately reach the nucleus and activate
tional Lymphoma Study Group, which proposed factors that initiate DNA synthesis (moving the cell
the World Health Organization/Revised European- from G1 to S phase of the cell cycle) or mitosis (moving
American Classification of Lymphoid Neoplasms from G2 to M). Oncogenes also support cell survival by
(REAL). The REAL classification includes genetic inhibiting apoptosis, or self-directed cell death. More
characteristics in addition to morphological tissue than 100 oncogenes have been identified in the human
architecture. With increasing ability to detect genome.
molecular characteristics of cells, including pat- Tumor suppressors include factors that control tran-
terns of gene expression, classification will con- scription, or the translation of genes required for cell
tinue to evolve.2,3 division. They also participate in repairing DNA damage
and in promoting apoptosis. Tumor suppressors slow
down or stop cell division by counteracting the move-
ment of the cell from G1 to S or G2 to M phase. These
MOLECULAR BASIS OF CANCER two points are therefore referred to as the G1 check-
point and G2 checkpoint in the cell division cycle. More
Cancer is caused by nonlethal mutations in DNA. The than 30 tumor-suppressor genes have been identified.
mutations affect two types of genes: oncogenes and In cancer cells, mutations in oncogenes are usually
tumor-suppressor genes. These genes control the cell gain-of-function mutations, resulting from amplifi-
division cycle and cell survival (Fig. 13.1). cation or translocation of DNA regions containing
Oncogenes promote cell division. Oncogenes include the genes or activating mutations that cause aberrant
cell membrane receptors that are bound by growth activity of the proteins. Mutations in tumor-suppressor
factors, hormones, and other extracellular signals. These genes are usually loss-of-function mutations, resulting
S
DNA synthesis
and chromosome
G1 replication
Cell growth
G2
Cell growth
M
FIGURE 13.1 The cell division cycle. After Mitosis
mitosis (M), there are two haploid (one diploid) and
complements of chromosomes (46 chromosomes) in cytokinesis
the G1 phase of the cell division cycle. DNA is rep-
licated during the S phase, resulting in four haploid
(two diploid) complements in the G2 phase. The
chromosomes are distributed to two daughter cells
at mitosis, with each receiving 46 chromosomes.
Cancer results when the cell division cycle proceeds
from G1 to S or G2 to M phase inappropriately.
in inactivation of the tumor-suppressor gene products. GENE AND CHROMOSOMAL MUTATIONS

These mutations may occur through deletion, translo- IN SOLID TUMORS
cation, or mutation of the genes. Molecular laboratory
testing aids in the diagnosis and treatment strategies for Molecular tests are routinely performed to aid in the
tumors by detecting abnormalities in specific tumor sup- diagnosis, characterization, and monitoring of solid
pressors or oncogenes. tumors. Some of these tests have been part of molecu-
lar pathology for many years. Others are relatively new
to the clinical laboratory. The methods applied to detect
ANALYTICAL TARGETS OF MOLECULAR TESTING the molecular characteristics of tumors are described in
previous chapters.
Although not yet completely standardized, several
tests are performed in almost every molecular pathol-
Human Epidermal Growth Factor
ogy laboratory. These tests assess tissue-specific and
Receptor 2, HER2/neu/erb-b2 1 (17q21.1)
tumor-specific targets. Tissue-specific targets are molec-
ular characteristics of the tissue from which a tumor HER2/neu was discovered in rat neuro-/glioblastoma
arose. The presence of DNA, RNA, protein, or other cell lines in 1985.1 Later it was found to be the same
molecules from these targets in abnormal amounts or gene as the avian erythroblastic leukemia viral onco-
locations is used to detect and monitor the presence of gene homolog 2, or ERBB2. Its gene product is a 185-kd
the tumor. For example, molecular tests are designed to cell membrane protein that adds phosphate groups to
detect DNA or RNA from cytokeratin genes in gastric tyrosines on itself and other proteins (tyrosine-kinase
cancer, carcinoembryonic antigen in breast cancer, and activity). This receptor is one of several transmem-
rearranged immunoglobulin or T-cell receptor genes brane proteins with tyrosine-kinase activity (Fig. 13.2).
in lymphoma. Although tissue-specific markers are It is very similar to a family of epidermal-growth-
useful, they are also expressed by normal cells, and factor receptors that is overexpressed in some cancers2
their presence does not always prove the presence (Fig. 13.3). In normal cells, this protein is required for
of cancer. cells to grow and divide. HER2/neu is overexpressed
In contrast, tumor-specific targets are not present in in 25% to 30% of human breast cancers, in which
normal cells and are, therefore, more definitive with overexpression of HER2/neu is a predictor of a more
respect to the detection of a tumor. Tumor-specific aggressive growth and metastasis of the tumor cells. It
genetic structures result from genome, chromosomal, or is also an indication for the use of anti-HER2/neu anti-
gene abnormalities in oncogenes and tumor-suppressor body drug therapy, which works best on tumors over-
genes that are associated with the development of the expressing HER2/neu. Herceptin therapy is indicated
tumor. Gene mutations and chromosomal translocations presently for women with HER2/neu-positive (HER2/neu-
are found in solid tumors, leukemias, and lymphomas. overexpressed) breast cancer.
Cell free nucleic acid or circulating tumor cells carrying Overexpression of the HER2/neu oncogene is per-
oncogenic mutations can be detected in blood and other formed by immunohistochemistry (IHC) using mono-
body fluids. Genome mutations, or aneuploidy, result in clonal and polyclonal antibodies to detect the HER2/
part from the loss of coordinated DNA synthesis and cell neu protein. The IHC test works best on fresh or frozen
division that occurs when tumor suppressors or onco- tissue. Fluorescent in situ hybridization (FISH), which
genes are dysfunctional. The following sections describe measures DNA and RNA of HER2/neu, is more reliable
procedures commonly performed in molecular pathol- than IHC, especially in older, fixed tissue,4 but is less
ogy laboratories; however, due to the rapid advances in readily available.
this area, the descriptions cannot be all-inclusive. The Southern, northern, and western studies have shown
discussion is divided into solid tumor testing and testing that HER2/neu gene amplification is highly correlated
for hematological malignancies. As will be apparent, with the presence of increased HER2/neu RNA and
however, some tests are applicable to both types of protein.5 In contrast to IHC, which measures increased
malignancies. amounts of HER2/neu protein, FISH directly detects
Outside of cell
Cysteine-rich
domain
Immunoglobulin-like
Transmembrane
domain
domain
Cell membrane
Kinase
domain
EGFR IGFR NGFR PDGFR FGFR VEGFR EPHR
Cytoplasm
FIGURE 13.2 Receptor tyrosine kinases include epidermal growth factor receptor (EGFR), insulin growth factor receptor (IGFR),
nerve growth factor receptor (NGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR),
vascular endothelial growth factor receptor (VEGFR), and ephrin receptor (EPHR). These molecules share similarities in that they
include a kinase domain, transmembrane domain, cysteine-rich domain, and immunoglobulin-like domain. The EPH receptor has
two fibronectin type III domains.
increased copy numbers of the HER2/neu gene in DNA are permanent, facilitating documentation and consul-
likely responsible for the increased protein. HER2/neu tations. Another bright-field imaging method, silver-
gene amplification occurs as a result of tandem dupli- enhanced in situ hybridization (SISH), introduced for
cation of the gene or other genetic events as the tumor the determination of HER2 status, has a high correlation
cells continue to divide. FISH testing for HER2/neu gene with FISH.7 Initially, in situ hybridization with chromo-
amplification requires a labeled probe for the HER2/neu genic detection by gene-specific probes was limited by
gene and a differently labeled control probe for the cen- high background and low signal intensity. Probes with
tromere of chromosome 17. For instance, a probe that repetitive sequences removed were designed to increase
spans the entire HER2/neu gene labeled in orange and specificity. Although FISH, SISH, and CISH are more
a probe that binds to the centromere of chromosome 17 accurate and less subjective methods than IHC, IHC is
labeled in green should yield two green signals, each faster, is less expensive, and allows the pathologist to
associated with an orange signal per nucleus. The copy assess target gene expression along with other visible
number of HER2/neu relative to centromere 17 indicates landmarks on the slide. Furthermore, protein overex-
whether HER2/neu is amplified (present in multiple pression (detectable by IHC) can occur without gene
copies on the same chromosome). Data are reported as a amplification (detectable by ISH). Some laboratories use
ratio of the number of HER2 signals to chromosome 17 IHC as an initial screening method and then confirm the
centromere signals. A ratio of more than 2 is considered results with ISH.
positive or amplified. The number of signals is enumer-
ated in 50 to 100 cells.
Epidermal Growth Factor Receptor,
Chromogenic in situ hybridization (CISH) is another
EGFR (7p12)
method that has been used to detect HER2/neu gene
amplification.5,6 Using a standard bright-field micro- Like HER2/neu, the epidermal growth factor receptor
scope, CISH technology also detects deletions, trans- gene (EGFR, ERBB1) is a member of the ERBB family
locations, or a change in the number of chromosomes. of growth factor receptors that also includes ERB3/HER3
An attractive feature of CISH is that the slide images and ERB4/HER4 (see Fig. 13.3). All of these proteins
Outside of cell Outside of cell Growth factor
EGF Heregulins NRG

TGF-␣ ? NRG 1-2 Heregulins
Tyrosine
kinase
receptor
Cell
membrane Cell Kinase
membrane domain
Cytoplasm
EGFR HER2 HER3 HER4
HER1 ERBB-2 ERBB-3 ERBB-4
ERBB-1
FIGURE 13.4 Upon binding by epidermal growth factor
Cytoplasm (EGF), the EGFR receptor in the cell membrane forms a dimer
with itself or with other members of the ERBB family of
FIGURE 13.3 The ERBB family of growth factor receptors receptors. The dimerization initiates a cascade of events, start-
includes the HER2 receptor and EGFR. The factors that bind ing with phosphorylation of the receptor itself, catalyzed by
to these receptors on the cell surface begin a cascade of events, the kinase domain.
including autophosphorylation and phosphorylation of other
proteins by the receptors. These factors include the epidermal
growth factor (EGF), human transforming growth factor alpha antibody drugs8 and methods approved by the U.S.
(TGF-α), heregulins, and neuregulins (NRGs). EGF, NRGs, Food and Drug Administration (FDA) for this appli-
and heregulins are small peptides that are active in the devel-
cation. Interpretation of the results of EGFR expres-
opment of various cell types, such as gastric mucosa, the heart,
and the nervous system.
sion testing and the predictive value of the test are not
always straightforward, however.8,9 Quantitative poly-
merase chain reaction (PCR) has also been proposed to
are located in the cell membrane and form dimers with assess EGFR gene copy number through increased gene
one another upon binding of growth factor from outside expression.10,11
the cell (Fig. 13.4). Binding of growth factors evokes Particular mutations in the kinase domain of the
tyrosine-kinase activity from intracellular domains of EGFR protein have proven better predictors of response
the receptors and initiates proliferation signals through to tyrosine-kinase inhibiting agents.12–15 These muta-
the cell cytoplasm. tions can be detected by a number of methods, includ-
EGFR is frequently overexpressed in solid tumors. ing sequence-specific PCR, single-strand conformational
Overexpression has been observed in a variety of tumor polymorphism (SSCP), and direct sequencing.13,16,17 The
types. For this reason, the EGF receptor has been an ever-expanding spectrum of clinically significant muta-
attractive target for the design of therapeutic drugs. tions has increased the use of next-generation sequenc-
Monoclonal antibodies were developed to block ligand ing (NGS) technology for this purpose.18,19
(growth factor) binding to the receptor. Agents were also Testing for predictors of response or prognosis, even
designed to inhibit the kinase activity of the receptor. with comprehensive sequencing, is complex because
The efficacy of these tyrosine-kinase inhibitors (TKIs) multiple clinical and genetic factors contribute to the
has been confirmed in clinical trials. response to targeted therapies and the natural course of
IHC analysis of EGFR protein expression, similar to the tumor. These include intronic polymorphisms in the
the testing for HER2/neu protein overexpression, was EGFR gene,20 expression of other components of the
assessed for correlation with response to monoclonal signal transduction pathway,21 or other tumor suppressors
such as p53.22 Also, multiple molecular and morpholog- are small proteins that bind to GTP in order to become
ical characteristics can be found in single tumors as a active. These small GTP-binding proteins include the
result of tumor heterogeneity.23,24 ras genes that receive signals from the cell surface pro-
teins and activate the initial steps of the MAPK cascade
(Fig. 13.6). Gain-of-function mutations in ras proto-
Kirsten Rat Sarcoma Viral Oncogene
oncogenes occur in many types of cancers.
Homolog, K-ras (12p12); Neuroblastoma ras,
N-ras (1p13); and Harvey Rat Sarcoma Viral
Oncogene Homolog, H-ras (11p15)
Advanced Concepts
Signals from extracellular stimuli, such as growth factors
or hormones, are transmitted through the cell cytoplasm Regulation of ras GTPase activity is controlled by
to the nucleus, resulting in cell proliferation or differen- rasGAP. Several other GTPase-activating proteins
tiation (Fig. 13.5). The mitogen-activated protein kinase (GAPs) besides rasGAP are important in signal
(MAPK) pathway is a cascade of phosphorylation events transduction. Two clinically important proteins of
that transduces growth signals from the cell membrane the GAP family of proteins are the gene product
to the nucleus. Critical components of this pathway of the neurofibromatosis type-1 (NF1) locus and
the gene product of the breakpoint cluster region
(BCR) gene. The NF1 gene is a tumor-suppressor
Growth factor
gene, and the protein encoded is called neurofi-
Receptor bromin. The BCR locus is rearranged in the Phil-
adelphia+ chromosome (Ph+) observed in chronic
myelogenous leukemias and acute lymphocytic
Mitogen
leukemias.
Normal cell growth

Mammals have three different ras genes that produce
four similar proteins, K-ras, N-ras, and H-ras. The Ras
proteins differ only in their carboxy termini, the end
No mitogen of the proteins that anchor them to the inner surface of
the cell membrane (Table 13.1 and Fig. 13.7). Because
Cell arrest or apoptosis
No mitogen plus TABLE 13.1 Four ras Proteins Synthesized

oncogene or From Three Genes, K-ras, N-ras, and H-ras*
tumor suppressor
gene mutation
Protein Modification Location
K-ras4A Farnesylation + palmitoylation ?
K-ras4B Farnesylation + polybasic Plasma

amino acids membrane
FIGURE 13.5 Normally, cells grow in the presence of nutri-
N-ras Farnesylation + palmitoylation Golgi
ents and factors that stimulate cell division (mitogens). Lack of
mitogen stimulation results in cell arrest, or apoptosis. If onco- H-ras Farnesylation + palmitoylation Golgi
gene or tumor-suppressor gene mutations stimulate aberrant
growth signals, cells grow in the absence of controlled *K-ras4A and K-ras4B arise from alternate splicing of transcripts of the same
stimulation. gene.
they bind and hydrolyze GTP for energy, the ras genes Mutations in K-ras are the most common oncogene
are members of a family of G-proteins. The GTP mutations in human cancers. The most frequently occur-
hydrolysis is regulated by GTPase-activating proteins ring mutations are located in codons 12, 13, 22, and 61
(GAPs). in exons 2 and 3 of the KRAS gene. Clinically significant
mutations may also be found in exon 4. These mutations
affect sequences coding for the GTP-binding domain of
the protein and throw the KRAS protein into a perma-
Outside of cell nently active state that does not require stimulation from
GTP hydrolysis. As a result, the RAS proteins harboring
Growth factor
these single-nucleotide substitutions remain constitu-
Activated tyrosine tively active in the GTP-bound form.
kinase receptor
Cell membrane KRAS mutations are highly correlated with tumor his-
tology and may predict the progress of tumorigenesis in
early-stage tumors. Furthermore, the presence of KRAS
GEF Ras GAP mutations may affect treatment strategy, especially
P (active)
with targeted therapies such as kinase inhibitors and
GTP P farnesyl-transferase inhibitors (KRAS protein is local-
Ras Ras
(inactive) (inactive) ized to the cell membrane through a farnesyl group).
GDP KRAS mutations are also a target of liquid biopsies, that
Raf is, analysis of tumor-specific mutations in blood.25
KRAS mutations are detected and identified by direct
sequencing. Site-specific methods such as pyrosequenc-
Ras ing have also been developed. Sequencing of selected
(active) exons of the KRAS and NRAS and downstream BRAF
GTP Raf genes have become a common method for mutation
P
analysis in lung, colon, thyroid, and skin cancer.26,27
MEK MEK Certain characteristics of tumor tissue, such as poor
cellularity, tumor heterogeneity, and previous chemo-
P
MAPk MAPk therapy treatments affecting tumor cells, confound the
Cytoplasm molecular diagnosis. These factors can be assessed and
noted during initial tissue collection and processing.28
Ewing Sarcoma, EWS (22q12)

Nucleus
A group of tumors arising from primitive neuroecto-
dermal tissue (PNET), Ewing sarcomas comprise a
family of childhood neoplasms referred to as the Ewing
family. Although immunohistochemical staining for the
cell surface protein HBA71 (p30/p32MIC2), a neuron-
FIGURE 13.6 Activation of membrane-bound K-ras is initi- specific enzyme, is helpful in the diagnosis of these
ated by activated receptors bound to mitogens (growth factors
tumors, no unique characteristics distinguish the differ-
or hormones). Active K-ras bound to GTP then initiates a
cascade of phosphorylation events that ends in the nucleus,
ent types of tumors that make up this group.
where transcription factors modulate gene expression. GDP/ Detection of specific translocations by cytogenetic or
GTP exchange on K-ras is modulated by GTPase-activating molecular methods is useful for diagnostic and prognos-
proteins (GAPs), guanosine nucleotide exchange factors tic accuracy (Table 13.2). Translocations involving the
(GEFs), and guanosine nucleotide dissociation inhibitors EWS gene at 22q12 (also called EWSR1 for EWS break-
(GDIs). point region 1) with the FLI-1 gene at 11q24, t(11;22)
Outside of cell
Cell
membrane
Farnesyl Palmitate
Cytoplasm Ras
FIGURE 13.7 Ras proteins are anchored to the cell

membrane through farnesyl groups (chemical structure
at bottom) and palmitoyl groups on the ras proteins. CH3 CH3 CH3
K-ras has only farnesyl groups, whereas N-ras and
H-ras have both farnesyl and palmitoyl groups. H3C C CH CH2 CH2 C CH CH2 CH2 C CH CH3
all samples to avoid false-negative results. These tests

TABLE 13.2 EWS Translocation Partners can be performed on tissue or liquid biopsies.34,35
Translocation Tumor
Synovial Sarcoma Translocation,
EWS-FLI-1 Ewing sarcoma, peripheral PNET (72%) Chromosome 18—Synovial Sarcoma
Breakpoint 1 and 2, SYT-SSX1, SYT-SSX2
EWS-ERG Ewing sarcoma, peripheral PNET (11%)
t(X;18)(p11.2;q11.2)
EWS-WT1* Desmoplastic small round cell tumor
A recurrent reciprocal translocation between chromo-
EWS-ATF1 Clear cell sarcoma some 18 and the X chromosome is found in synovial
sarcoma, a rare type of cancer of the muscle, fat, fibrous
*The WT1 gene is also associated with Wilms’ tumor (WT), one of the tissue, blood vessels, or other supporting tissue of the
common solid tumors of childhood, accounting for 8% of childhood cancers.
Several genes or chromosomal areas affect the development of WT: WT1
body. Synovial sarcoma accounts for 8% to 10% of all
at 11p13, WT2 at 11p15.5, WT3 at16q, WT5 at 7p15-p11.2, and WT4 at sarcomas and occurs mostly in young adults. About 80%
17q12-q21. of cases have the t(X;18) translocation.
The t(X;18) translocation fuses the synovial sarcoma
translocation, chromosome 18 gene (SS18 or SYT) with
(q24;q12) are present in 85% of Ewing sarcomas. either of two related genes, synovial sarcoma translo-
Another translocation between EWS and the ERG gene cated to X (SSX1 and SSX2), on the X chromosome.
at 21q22 is present in 5% to 10% of Ewing sarcomas. There are five SSX variants, SSX1, SSX2, SSX3, SSX4,
Other partners for the EWS gene, such as ETV1 at 7p22, and SSX5. With rare exceptions, only SSX1 and SSX2 are
E1AF at 17q12, and FEV at 2q33, are present in fewer fused to SYT in the t(X;18) translocation.36 The fusion
than 1% of cases.29 The occurrence of these rearrange- gene acts as an aberrant transcription factor, with both
ments was first revealed by cytogenetics30,31 and then by activation and repression functions from the SYT and
PCR methods.32,33 SSX portions, respectively.
Laboratory testing at the molecular level includes The t(X;18) translocation is detected by FISH or
detection of the tumor-specific translocations by reverse RT-PCR.37 In the latter method, total RNA reverse-
transcriptase polymerase chain reaction (RT-PCR; transcribed to cDNA is amplified with primers specific
Fig. 13.8). Positive results are revealed by the presence for SSX and SYT genes. In a semi-nested PCR version of
of a PCR product. Negative specimens will not yield a this procedure, the SSX primer used in the first round is a
product. As with all assays of this type, an amplification consensus primer for both SYT-SSX1 and SYT-SSX2. After
control, such as GAPDH or 18S RNA, must accompany the first amplification, SSX1- and SSX2-specific primers
EWS gene FLI1 gene
Chromosome 22 Chromosome 11
Translocation
EWS gene FLI1 gene
Fusion gene
FIGURE 13.8 RT-PCR is used to detect the

t(11;22) mutation in Ewing sarcoma. One
Fusion mRNA isolated
primer is designed to hybridize to the EWS
from cells
gene on chromosome 22 and one primer to the
Reverse transcriptase
Primer FLI1 gene on chromosome 11. If the transloca-
tion has occurred, the resulting fusion tran-
script isolated from the tumor cells will yield
cDNA cDNA that is amplifiable with the EWS and
Primer FLI1 primers.
discriminate between the two translocation types. The DNA-binding motifs homologous to the forkhead tran-
PCR products are detected by agarose gel electrophore- scription factor first discovered in the fruit fly, Drosoph-
sis and ethidium bromide staining. This method can be ila. PAX-FKHR translocations have been observed in all
performed on fresh, frozen, or fixed tissue, depending on subtypes of RMS but are more characteristic of ARMS.
the condition of the specimen RNA. Furthermore, PAX7-FKHR, t(2;13), is associated with
better outcomes than PAX3-FKHR, t(1;13). Mutations
in the PAX3 gene are also found in Waardenburg syn-
Paired Box-Forkhead in
drome, a congenital auditory pigmentary syndrome.39,40
Rhabdomyosarcoma, PAX3-FKHR,
The majority of ARMS displays the t(2;13) transloca-
PAX7-FKHR, t(1;13), t(2;13)
tion, with the t(1;13) variant present with one-third the
Rhabdomyosarcoma (RMS) is the most common soft frequency as t(2;13). Both translocations are detected by
tissue sarcoma of childhood, accounting for 10% of all FISH, RT-PCR, quantitative polymerase chain reaction
solid tumors in children. In addition to alveolar rhabdo- (qPCR), and RNA sequencing.41–43
myosarcoma (ARMS), there are two additional histolog-
ical forms of RMS: embryonal (RMS-E) and primitive
Tumor Protein 53, TP53 (17p13)
(RMS-P). Although histological classification of RMS
is sometimes difficult, accurate diagnosis is impor- Mutations in TP53 are found in all types of cancer, and
tant for the management and treatment of this malig- about 50% of all cancers have TP53 mutations. The gene
nancy because ARMS has a worse prognosis than other product of TP53, p53, is a 53,000-dalton DNA-binding
subtypes. protein that controls the expression of other genes. Nor-
Translocations involving the Forkhead in the Rhab- mally, p53 participates in the arrest of cell division in
domyosarcoma gene (FKHR, also called FOXO1A) and the event of DNA damage. The arrest in the G1 phase
the paired box genes (PAX3 and PAX7) are frequently of the cell cycle allows repair enzymes to correct the
found in ARMS.38 The chimeric genes resulting from DNA damage before DNA synthesis begins. Once the
the translocations encode transcriptional activators with damage is repaired, p53 protein is removed by binding
to another protein, MDM2, and through degradation.

When p53 is not functional, replication proceeds on TABLE 13.3 Sources of Potential Inaccuracies
damaged templates, resulting in the potential for further of p53 Mutation Analysis
genetic abnormalities. Also, the mutant protein does not
degrade properly and accumulates in the cell nucleus Method False Positive False Negative
and cytoplasm. Due to the frequency and ubiquity of its IHC Staining of Deletions or mutations
mutations, mutated p53 protein is a potential therapeutic normal p53 in p53 that remove
target.44 Small molecules that can reactivate mutant p53 protein Ab binding epitopes;
protein have been tested in clinical trials. promoter mutations
Several studies have shown that mutated TP53 in SSCP Alternate Less than 5% mutant
tumor tissue is also an indicator of poor prognosis in conformers; cells in specimen;
breast cancer, lung cancer, colon cancer, leukemia, and silent DNA mutations outside of
other types of cancers. The significance of TP53 status as polymorphisms the exons screened
a predictor of decreased survival time or tumor relapse, Sequencing PCR mutagenesis; Less than 10% mutant
however, became controversial.45 Part of the controversy high background cells in specimen;
arose from the different methods used to detect TP53 mutations outside of
mutations. A common method was the detection of the sequenced area
stabilized mutant protein by IHC. Several monoclo-
nal antibodies directed at different epitopes in the p53
protein were used for this purpose. Because normal p53
protein is transient, significant staining (+2 or above detected, further analysis of relatives requires targeting
on a scale of 0 to +4) of p53 is considered positive for only that mutation.
the mutation. Use of microarrays or panel sequencing
to screen expression of multiple genes along with TP53
Ataxia Telangiectasia Mutated Gene, ATM
was proposed as a more accurate method for predicting
(11q22)
survival than either IHC or mutation analysis of TP53
alone. Methods that include sequencing of the entire Predisposition to cancer is one symptom of the neuro-
TP53 coding region on cDNA, in combination with logical disease ataxia telangiectasia (AT). AT occurs in
IHC, is another accurate approach. at least 1/40,000 live births. This disease is caused by
SSCP and direct sequencing of microdissected mutations in the ATM (A-T mutated) gene on chromo-
tumor tissue are other methods often used to detect some 11. ATM mutations are also present in some types
TP53 mutations. SSCP methods cover selected exons 5 of leukemias and lymphomas. Carriers of the autosomal-
to 8 or 4 to 9 of the TP53 gene because these exons recessive mutations in ATM are at increased risk for
encode the regions involved in DNA binding and developing leukemia, lymphoma, or other types of
protein–protein interactive functions of the p53 protein. cancers.
Sequencing methods can include the entire gene. In The ATM gene product is a member of the phospha-
early studies, mutations detected by IHC were not tidylinositol-3 kinase family of proteins that respond
always consistent with mutations found by direct DNA to DNA damage by phosphorylating other proteins
analysis.46,47 There are several explanations for these involved in DNA repair and/or control of the cell cycle.
discrepancies (Table 13.3). The ATM protein participates in pausing the cell cycle
In addition to screening for somatic alterations, at the G1 or G2 phase to allow completion of DNA
mutation analysis of TP53 is also performed to aid in repair.
the diagnosis of Li–Fraumeni syndrome, a cancer-prone Direct DNA sequencing is the method of choice
condition caused by inherited mutations in the p53 gene. for detection of ATM mutations, especially in family
In this case, normal tissue will be heterozygous for members of carriers of previously identified mutations.
the mutation, removing the challenge of isolating pure Other methods, such as SSCP, have been used. A func-
samples of tumor tissue. Once an inherited mutation is tional test for the repair of double-strand breaks induced
by irradiation was developed for ATM. For this assay,

exponentially growing cells were irradiated, followed Advanced Concepts
by the addition of Colcemid to inhibit spindle forma-
tion. The cells were harvested for Giemsa staining, and PolyADP-ribosyl transferase modifies nuclear pro-
the karyotypes were examined. The ratio of aberrations/ teins by polyADP-ribosylation. The modification
cell was calculated from the number of chromatid and is involved in the regulation of important cellular
chromosome breaks (counted as one breakage event) in processes, including the molecular events involved
addition to dicentric chromosomes, translocations, ring in the recovery of cells from DNA damage. In addi-
chromosomes, and chromatid exchange figures (counted tion, this enzyme may be the site of mutation in
as two breakage events).48 More recent measures of Fanconi anemia and may participate in the patho-
DNA damage utilize chromogenic reporters.49,50 physiology of type 1 diabetes. Fanconi anemia is a
If a mutation is identified in a patient with ataxia tel- genetically heterogeneous recessive disorder char-
angiectasia, other family members may be tested for the acterized by defective DNA repair. Mutations in
presence of the same mutation. Presence of a mutation at least 15 genes can cause Fanconi anemia. The
in family members identifies those with an increased Fanconi anemia complementation group (FANC)
risk of AT. Heterozygous carriers of an ATM mutation includes FANCA, FANCB, FANCC, FANCD1
may also be at increased risk for mantle cell lymphoma, (=BRCA2), FANCD2, FANCE, FANCF, FANCG,
B-cell lymphocytic leukemia, or T-cell prolymphocytic FANCI, FANCJ, FANCL, FANCM, and FANCN
leukemia. (=PALB2). These proteins are related as compo-
nents of the nuclear protein complex.
Breast Cancer 1 Gene, BRCA1 (17q21),
and Breast Cancer 2 Gene, BRCA2 (13q12)
Approximately 5% of breast cancers result from inher-
ited gene mutations, mostly in the breast cancer genes SSCP, protein truncation tests, chromosome breakage
BRCA151,52 and BRCA2.53,54 Women who carry a muta- tests, and other procedures have been used to screen for
tion in BRCA1 have a 60% to 80% lifetime risk of breast mutations in the BRCA genes. The method used for clin-
or ovarian cancer. Men carrying a mutation, especially ical applications, however, is direct sequencing. Three
in BRCA2, have a 100-fold increased risk of breast mutations, 187delAG and 5382insC in BRCA1 and
cancer compared with men without a mutation, as well 6174delT in BRCA2, occur frequently in particular ethnic
as increased risk of colon and prostate cancer. Both men populations. These known mutations can be detected
and women can transmit the mutation to subsequent by several targeted assays, including sequence-specific
generations. PCR and allele-specific oligomer hybridization.
The BRCA1 gene product has a role in embryonic Just as with any genetic analysis, testing for BRCA1
development, and both BRCA1 and BRCA2 gene prod- and BRCA2 mutations requires patient counseling and
ucts are involved in DNA double-strand break repair education. The significance of a BRCA mutation test
by homologous recombination. Defects in homologous will depend on several factors, including penetrance of
recombination repair (HRR) are associated with muta- the gene mutations. Furthermore, if a mutation is not
tions in repair genes, including BRCA1/BRCA2, ATM, detected in the coding sequences of the genes, the pos-
ATR, PALB2, RAD51, CHEK1, and CHEK2, as well as sibility of mutations in the noncoding regions cannot be
loss of BRCA1 expression through promoter methylation ruled out.
or overexpression of the BRCA2 transcriptional repres-
sor EMSY.55 Poly(ADP-ribosyl)transferase or PARP is
Von Hippel–Lindau Gene, VHL (3p26)
also part of the repair process, and therapeutic agents
inhibiting its activity in combination with other repair Benign blood vessel tumors in the retina were first
gene inhibitors have shown efficacy against BRCA- reported by Eugen von Hippel, a German ophthal-
associated cancers.56 mologist, in 1895. In 1926, Arvid Lindau, a Swedish
pathologist, further noted that these retinal tumors were

V-Ros Avian UR2 Sarcoma Virus Oncogene
linked to tumors in the blood vessels in other parts of
Homolog 1 (ROS1) Proto-Oncogene
the central nervous system, sometimes accompanied by
(6q22.1) and Rearranged During
cysts in the kidneys and other internal organs, and that
Transfection (RET) Proto-Oncogene (10q11)
the condition was heritable. The Von Hippel–Lindau
syndrome (VHL) is now recognized as a genetic con- The ROS1 oncogene, coding for a membrane receptor
dition involving the abnormal growth of blood vessels tyrosine kinase, is rearranged in a variety of human
in organs, especially those that are particularly rich in cancers. The resulting fusion protein contains a consti-
blood vessels. It is caused by mutations in the VHL tutively active ROS1 kinase domain and drives cellu-
gene, which is located on the short arm of chromo- lar proliferation. ROS1 is rearranged in 1% to 3% lung
some 3. Normally, VHL functions as a tumor-suppres- adenocarcinomas.
sor gene, promoting cell differentiation. VHL syndrome The RET proto-oncogene is located on the long arm
is a predisposition for renal cell carcinoma and other of chromosome 10 (10q11). Its gene product is a mem-
cancers.57 brane tyrosine kinase that participates in sending cell
Deletions, point mutations, and splice-site mutations growth and proliferation signals to the nucleus. The first
have been described in patients with VHL. In addition, intron in the gene covers about 24 kb, with exons 2 to
cases of renal cell carcinoma and tumors of the adrenal 20 contained in the remaining 31 kb. This general struc-
gland are accompanied by somatic mutations in the ture of a large first intron with small exons is character-
VHL gene. Direct sequencing is the preferred method of istic of tyrosine-kinase receptors, such as the receptors
testing for VHL gene mutations. for the KIT, EGFR, and platelet-derived growth factor
(PDGF) genes.
The RET gene is an example of how different muta-
V-myc Avian Myelocytomatosis Viral-
tions in the same gene result in different diseases. Trans-
Related Oncogene, Neuroblastoma-
locations that result in overexpression of RET are found
Derived, MYCN or n-myc (2p24)
in thyroid papillary carcinomas. Point mutations that
Myc family proteins, activated by mitogen signals, activate RET (also called MEN2A) are found in inherited
increase the expression of several genes through inter- multiple endocrine neoplasia (MEN) syndromes, a group
actions with conserved cis elements (Myc-boxes) and of diseases resulting in abnormal growth and function
transcriptional coactivators. The Myc family includes of the pituitary, thyroid, parathyroid, and adrenal glands.
the genes c-myc, n-myc, and l-myc. They are basic helix- In contrast, loss-of-function mutations in the RET gene
loop-helix transcription factors.58 are found in Hirschsprung disease, a rare congenital lack
The c-myc oncogene (8q24.21) is the most frequently of development of nerve cells in the colon that results
expressed myc. The c-myc gene is amplified in breast in colonic obstruction. Mutations have been reported in
and ovarian cancer, lymphomas, and leukemias. Myc about 50% of congenital cases and 20% of sporadic cases
expression is regulated by transcription factors specific of this disorder. Because about 16% of children with con-
to each cancer type. Increased Myc expression is a nega- genital central hypoventilation syndrome (CCHS) have
tive prognostic marker. Expression of l-myc (1p34.2) can Hirschsprung disease, RET mutations were also sought
induce differentiation in cultured cells.59 Amplification in CCHS, but most of the mutations detected were deter-
of l-myc is associated with oral cancer. mined as polymorphic variants.62
The n-myc gene on the short arm of chromosome 2 Detection of RET gene mutations can aid in the diag-
(2p24) is amplified in cases of neuroblastoma and reti- nosis of MEN diseases. Clinical testing targets, mainly
noblastoma. n-myc is an oncogene that is counteracted exons 10, 11, and 16, are where most reported mutations
by the tumor-suppressor gene neurofibromatosis type 1 have been found.
(NF1). Myc gene amplification (or protein expression) RET and ROS1 rearrangements are detected by IHC,
is detectable by IHC, FISH, sequencing, or array analy- FISH, or sequencing.63 RET alterations occur in about
sis.60 Transcription of n-myc may also be measured using 1% of lung cancers, and both RET and ROS1 are thera-
qPCR.61 peutic targets.64
Anaplastic Lymphoma Receptor Tyrosine to cancer in this syndrome is caused by mutations in the
Kinase (ALK) Proto-Oncogene, 2p23.1 mismatch repair (MMR) protein complexes, including
mutS homologs, MSH2, MSH6, mutL homolog MLH1,
ALK is a receptor tyrosine kinase, classified in the and the human postmeiotic segregation hPMS2.66 These
insulin receptor superfamily. The ALK gene has been complexes are responsible for correcting replicative
found to be rearranged, mutated, or amplified in tumors errors and mismatched bases in DNA. The MMR system
of various types, including lymphomas, neuroblastoma, was originally discovered in bacteria (Escherichia coli)
and non–small-cell lung cancer. Chromosomal rear- and further studied in yeast (Saccharomyces cerevisiae).
rangements are the most common genetic alterations in Similar (homologous) genes were subsequently identi-
this gene, with multiple fusion gene partners, including fied in humans and named after the bacterial and yeast
the Echinoderm Microtubule-Associated Protein-Like genes (Table 13.5). The protein complex binds to mis-
4 gene (EML4; 2p21) and others. ALK gene rearrange- matched bases in the DNA double helix or loops formed
ments are detected by FISH analysis and sequencing. by replicative errors. The repair is methyl-directed. At
the end of S phase (DNA replication), the system rec-
V-Kit Hardy-Zuckerman 4 Feline Sarcoma ognizes errors in the newly synthesized (unmethylated)
Viral Oncogene Homolog, KIT, c-KIT (4q12) daughter strand and uses the template strand, which is
methylated, as a guide for repair (Fig. 13.9).
KIT protein is a transmembrane receptor with tyrosine Included in the types of DNA lesions that are repaired
kinase activity. Mutations in this gene are associated by this system are replication errors (RERs) caused by
with gastrointestinal stromal tumors (GISTs), mast cell slippage between the replication apparatus and the DNA
disease, and AML. KIT activation has oncogenic activ- template (Fig. 13.10). RERs occur especially in micro-
ity. Targeted therapeutics have been used to treat patients satellites where one to three nucleotides are repeated
with melanoma and GIST. Increased KIT activity can in the DNA sequence. If the errors remain in the DNA
result from amplification, overexpression or missense until the next round of replication, new alleles will arise,
mutations. IHC is used to detect increased KIT protein, generating increasing numbers of alleles for the locus,
and sequencing is used for detection of the missense or microsatellite instability (MSI). In contrast, a stable
mutations. locus will retain the same alleles through many rounds
of replication. Microsatellite slippage occurs about every
Other Molecular Abnormalities 1,000 to 10,000 normal cell divisions, most of which are
repaired in normal cells. Dysfunction of one or more
Increasing numbers of molecular abnormalities are being components of the MMR system will result in MSI, an
used to aid in the diagnosis and monitoring of solid increase in the number of alleles due to loss of repair.
tumors. Some examples of potential diagnostic targets The majority of MMR mutations in Lynch syndrome
are shown in Table 13.4. As molecular aberrations in are found in the MSH2 and MLH1 genes. Mutations in
oncogenes and tumor-suppressor genes are identified, hPMS2 account for 9% of MMR variants, compared
molecular analysis becomes more important in their with 39% MLH1 and 33% MSH2 and 19% MSH6 (see
rapid and accurate detection. http://www.insight-database.org/genes). Mutations in
hPMS1 and MSH3 are rare. Although direct sequenc-
ing of the affected genes is definitive and identifies the
Microsatellite Instability
specific mutation in a family, the test may miss muta-
Lynch syndrome, or hereditary nonpolyposis colorectal tions outside of the structural gene sequences or in other
cancer (HNPCC),65 is an inherited cancer predisposition genes. Screening for MSI normal MMR proteins by IHC
syndrome, accounting for 3% to 5% of colon cancers and is often the first step in MSI analysis.
3% of endometrial cancers. There is also an increased Lack of staining of the normal protein is evidence of
risk of developing other types of cancers, including loss of MMR function. Because this loss of MMR gene
cancer of the stomach, breast, ovary, pancreas, prostate, function causes MSI, MSI can be used to screen indi-
urinary tract, liver, kidney, or bile duct. Predisposition rectly for mutations in the MMR genes. With an inherited
TABLE 13.4 Molecular Abnormalities in Some Solid Tumors
Gene Location Mutation Detection Method Associated Disease
Adenomatous polyposis 5q21 5q deletion, t(5;10) Southern blot, FISH, Familial adenomatous polyposis of
of the colon sequencing, SSCP the colon
Retinoblastoma (Rb, RB1) 13q14.1 13q deletion, t(X;13) Southern blot, FISH, Retinal neoplasm, osteosarcoma
sequencing
MET proto-oncogene, 7q31 Missense mutations Sequencing Renal carcinoma

hepatocyte growth factor
receptor
KIT proto-oncogene, stem 4q12 Missense mutations Sequencing Gastrointestinal stromal tumors
cell factor receptor (SCFR)
Folliculin (FLCL, BHD) 17p11.2 Insertions, deletions Sequencing Birt–Hogg–Dube syndrome (hair
in a C8 tract in exon follicle hamartomas, kidney tumors)
11
Fumarate hydratase 1q42.1 Frameshift Sequencing Hereditary leiomyomatosis, renal

mutations cell cancer
Activin A receptor (ALK4) 12q13 Gene fusion Pancreatic carcinoma, lung cancer
Epidermal growth factor 7p12 Missense mutations, SSP-PCR, SSCP, Lung cancer, glioblastoma
receptor (EGFR) deletions Sequencing
Avian erythroblastic 17q21 Amplification Immunohistochemistry, Breast cancer

leukemia viral oncogene FISH
homolog (ERBB-2, HER2)
Platelet-derived growth 4q12 Deletion Immunohistochemistry, Idiopathic hypereosinophilic

factor alpha (PDGFRa) FISH, CISH syndrome
TABLE 13.5 Genes of the MMR System
Human Gene Bacterial Gene Function
MSH2 MutS Single mismatch, loop repair
MSH3 MutS Loop repair
MSH4 MutS Meiosis
MSH5 MutS Meiosis
MSH6/GTBP MutS Single mismatch repair
MLH1 MutL Mismatch repair
hPMS2 MutL Mismatch repair (postmeiotic segregation in yeast)
hPMS1 MutL Mismatch repair (postmeiotic segregation in yeast)

mutation in one copy of an MMR gene, somatic muta- alleles in the tumor tissue compared with that in the
tion of the remaining copy will result in the MSI pheno- normal tissue after PCR amplification of microsatellite
type in tumor cells. MSI, therefore, will be apparent in loci and gel electrophoresis (Fig. 13.11) or capillary gel
the tumor where both functional copies of the gene have electrophoresis (Fig. 13.12). The detection of instability
been lost but not in normal tissue that retains one normal (more bands or peaks in the tumor tissue compared with
copy of the gene. To perform MSI analysis, therefore, the normal tissue) is strong evidence for Lynch syn-
normal and tumor tissue from the patient must be com- drome. The National Cancer Institute has recommended
pared. MSI is apparent from the increased number of that screening two mononucleotide-repeat loci, BAT25
and BAT26, and three dinucleotide-repeat loci, D5S346,
D2S123, and D17S250, is sufficient for determination of
MSI.67 Alternate markers have been proposed, and some
MutL PMS2 laboratories test additional loci to ensure amplification
of at least five loci and address discordant or borderline
MutH MutS
samples.68 Further, mononucleotide-repeat structures
may be more sensitive markers for MSI than dinucle-
CH3 CH3 otide repeats, so some laboratories prefer to use only
Exonuclease, mononucleotide-repeat loci.
SSB, If at least two of the five, or if more then 30% of
Helicase
loci show instability, the specimen is classified as having
high instability (MSI-H). Tumors showing MSI in one
or a minority of loci tested are classified as low instabil-
CH3 CH3
ity (MSI-L). If no MSI is detected in the loci tested, the
Polymerase, tumor is stable (MSS). Initially, MSI-L and MSS tumors
Ligase are interpreted as microsatellite stable, and MSI-H
tumors are considered microsatellite unstable. More
recent clinical correlations suggest that MSI-L may have
specific implications.69 MSI-H is reported as MSI unsta-
CH3 CH3 ble, with an increased likelihood that the patient has
Lynch syndrome.69 If MSI is discovered, the inherited
FIGURE 13.9 The mismatch repair system recognizes a mis-
match (top) in the newly synthesized unmethylated strand of mutation can be confirmed by direct sequencing of the
DNA. The complex of proteins recruits exonucleases, sin- MMR genes.
gle-strand DNA-binding proteins (SSB), and helicases to Sequencing will not detect epigenetic silencing of the
remove the erroneous base (center). Polymerases and DNA MLH1 gene, which can also result in MSI. A separate test
ligase then replace the missing bases. for MLH1 promoter methylation by bisulfite sequencing
…TTTTTTT…
…AAAAAAA…
…TTTTTTT…
Replication …TTTTTT…
…AAAAAAA… error …AAAAAA…
…T TTTTT…
…A
AAAAAA… …TTTTTTT…
…AAAAAAA…
FIGURE 13.10 Replication errors result from slippage during DNA replication. If the error is not repaired, the next round of
replication will create a new allele (top right) of the original locus. Additional uncorrected errors will produce more alleles.
N = Normal or methylation-specific PCR may also be performed.70

T = Tumor Testing for deletion of the EPCAM gene by sequenc-
N T N T N T
ing or multiplex ligation-dependent probe amplification
(MLPA) is another approach to assess linked deletion of
MMR loci.71 The choice of methodology used will vary
among cases. The cost of testing increases with IHC,
PCR, and sequencing, respectively.72
Loss of Heterozygosity
Unstable loci (MSI)

Increased risk of cancer is a concern for members of
families carrying cancer-predisposition mutations. These
FIGURE 13.11 Microsatellite instability (MSI) is detected mutations are recessive regarding the cancer or malig-
by increased alleles compared with stability at the same locus. nant cell phenotype but dominant regarding the cancer
risk. Therefore, relatives of patients with suspected
inherited conditions are tested for purposes of cancer
surveillance and family planning.
Once the familial gene mutation is identified in the
patient or proband, targeted tests for the mutation in
other family members are performed. In an inherited
N condition such as Lynch syndrome or a history of breast
and ovarian cancer (HBOC), blood samples are sufficient
for mutation analysis. In tumor cells, further testing for
T loss of heterozygosity (LOH) may be performed. LOH
reveals the loss of the “good allele” at a locus, uncov-
ering the homologous locus with a recessive mutation.
LOH can be detected by PCR amplification of hetero-
N zygous STR or variable-number tandem repeat (VNTR)
loci closely linked to the disease gene.73 Amplification
of loci in tumor cells with LOH will reveal a loss of
T
the allele linked to the normal allele of the gene when
compared with the mutant allele (Fig. 13.13). Compar-
ing peak heights in normal (N) and tumor (T) tissues,
the formula for LOH is as follows:
N
(peak height of normal allele in N
peak height of normal alleele in T)
( peak height of mutant allele in T
T peak height of mutant allele in N)
A ratio of less than 0.5 or more than 2 indicates LOH.
Alternately, LOH is assumed if the peak height of the
normal allele in the tumor is less than 40% of the height
FIGURE 13.12 MSI detected by capillary gel electrophore-
sis. DNA from the tumor (T) is compared with DNA from of the normal allele in the normal DNA. Technical arti-
normal cells from the same patient (N). Increased alleles in the facts such as allelic dropout or PCR bias complicate
tumor scans reveal instability at those loci (top four scans). the interpretation. When analyzing tumor tissue, it may
Stable loci look the same in normal and tumor tissue (bottom be necessary to test more than one area of a tumor to
two scans). confirm LOH.
Homologous chromosomes
Normal
Normal Mutant
allele allele
Fluorescence
STR Diseased gene
Normal
Tumor
Normal Mutant
allele allele
FIGURE 13.13 Loss of heterozygosity (LOH) is
Fluorescence
detected by PCR and capillary electrophoresis of hetero-

zygous STR loci linked to disease genes. A deletion or
loss of the normal allele uncovering a recessive mutant
allele is identified by the loss of the STR linked to the
normal gene (right).
comparative genomic hybridization (CGH) array tech-

Liquid Biopsy
nology (e.g., digital karyotyping), with sensitivities of
Solid tumors release cells and nucleic acids into cir- 0.2% to 2%. They can detect copy-number variations
culation. This may have a role in metastatic cancers, and structural abnormalities. Plasma DNA is the most
which grow in multiple body sites. As the application frequent source material. Plasma RNA has seen less
of molecular testing advances, analysis of tumor cells clinical application but has the potential to provide addi-
has become increasingly informative. Surgical biop- tional information on tumor gene expression and tissue-
sies are commonly used for molecular analyses and are specific gene expression. The latter might be used to
preferable to surgical resections in some cases. With the determine the tissue of origin of tumors as well.75
development of efficient nucleic acid isolation methods Urine has also been a source of genetic information
and highly sensitive analyses, tumor cells and DNA in from cfDNA. Urine analysis has been applied to the
body fluids have become a valuable source of informa- detection of acquired mutations in lung cancer after
tion without the cost and comorbidity of surgical biop- treatment with targeted therapies.76,77
sies. The liquid biopsy is usually from blood plasma Circulating tumor cells (CTCs), first reported in
or serum or urine but may also be done on other body 1869, were considered to be a mechanism of tumor
fluids. There are two approaches to liquid biopsies: cell metastases from one tissue site to another. Although
free or circulating free nucleic acids (cfDNA or cfRNA), low in number, they can travel in clusters of 2 to more
exosomes (cell-derived vesicles carrying nucleic acid or than 50 cells.78 There are several technologies for the
other molecules) and circulating tumor cells (CTC). isolation of CTCs. Antibody capture, using immobilized
Circulating nucleic acid analysis can be classified as antibodies to epithelial cell–specific membrane proteins,
targeted or untargeted.74 Targeted procedures are directed such as the epithelial cell adhesion molecule (EpCAM),
at specific mutations using sequencing or PCR methods is most frequently used. Alternatively, negative deple-
specifically designed for those mutations or rearrange- tion of leukocytes can also be performed. Cell morphol-
ments. These methods can reach high sensitivity (1% ogy and physical characteristics such as size (epithelial
to 0.001%) and provide an estimation of mutant allele cancer cells are larger than leukocytes) or density may
frequency. Untargeted methods assess whole exomes be used for selection. Once the tumor cells are enriched,
or, more frequently, gene panels by sequencing and they may be stained and other characteristics measured.
Extracted DNA is tested using allele-specific PCR or intrachromosomal rearrangements in B and T lympho-
other sensitive methods. Gene-expression profiling and cytes, and the abnormal interchromosomal transloca-
translocations may be found in extracted cDNA made tions that can occur in any cell type.
from extracted RNA.
cfDNA and CTC are also sources of epigenetic V(D)J Recombination
information. Methylation patterns specific to tissues or
To enhance antibody diversity, lymphocytes undergo
tumor state can be assessed using sequencing, meth-
normal genetic rearrangement of immunoglobulin (Ig)
ylation-specific PCR, and methylation arrays. miRNA
heavy- and light-chain genes and T-cell receptor genes
can be isolated from plasma as well. Although not yet
(Fig. 13.14). The gene rearrangement process is a
part of routine clinical testing, the presence, absence,
series of intrachromosomal recombination events medi-
or expression levels of particular miRNA species may
ated by recombinase enzymes that recognize specific
provide information on prognosis or tumor state. Just as
sequences flanking the gene segments. This process
with DNA variants, annotated databases will provide a
occurs independently in each lymphocyte, so that a
basis for interpretation of the significance of epigenetic
diverse repertoire of antibodies is available to match any
analyses.
random invading antigen.
MOLECULAR ANALYSIS OF LEUKEMIA Immunoglobulin Heavy-Chain Gene

AND LYMPHOMA Rearrangement in B Cells
Each antibody consists of two heavy chains and two
Gene Rearrangements
light chains. The gene encoding the immunoglobulin
Gene rearrangements analyzed for hematological ma- heavy chain is located on chromosome 14. The unrear-
lignancies include V(D)J recombination, the normal ranged or germline configuration of the immunoglobulin
Mature
Early B-cell precursors Pre-B cell B cell
plasma cell
IgH GR IgH GR ± IgL GR IgH + IgL GR
Lymphoid
Early thymocytes Common
stem cell
thymocytes
Cytotoxic T cell
TCR and GR TCR and GR
Helper T cell
FIGURE 13.14 Gene rearrangements (GRs) are normal processes that occur in B and T lymphocytes as they mature from lym-
phoid stem cells. The genes coding for immunoglobulin heavy and light chains (IgH and IgL, respectively) begin the rearrange-
ment process in early B cells and pre–B cells. The T-cell receptor (TCR) genes rearrange in the order δ, γ, β, and α chains.
Germline heavy-chain gene alleles is referred to as allelic exclu-

L VH1 L VHN DH JH C sion. The rearrangement on the other chromosome will
proceed if the first rearrangement fails or is not produc-
tive. The recombination events of the gene rearrange-
ment are mediated by recombination activating genes,
RAG1 and RAG2. These genes code for enzymes that
Rearranged recognize short recombination signal sequences in the
DNA, where they form a complex that initiates the
cutting and re-ligation of the DNA.
When the DNA is cut in the process of gene rear-
L V DJ C rangement, terminal deoxynucleotidyl transferase may
add nucleotides at the V–D–J junctions, further diver-
FIGURE 13.15 The immunoglobulin heavy-chain gene on sifying the coding sequences of individual antibody
chromosome 14 consists of a series of variable (V), diversity genes. After the V(D)J rearrangement occurs, another
(D), and joining (J) gene segments (germline configuration). enzyme, activation-induced deaminase (AID), further
The V segments are accompanied by a short leader region (L). changes the DNA sequence of the variable region. These
One of each type of segment, V, D, and J, is selected and com- variable regions are said to have undergone somatic
bined by an intrachromosomal recombination event, first D
hypermutation.
and J, and then V and D. The C (constant) segments are joined
through splicing or a secondary recombination event, class
switching. Advanced Concepts
Chronic lymphocytic leukemia (CLL) arising
heavy-chain locus consists of a series of gene segments
from cells harboring the “mutated” variable region
or repeated exons coding for the functional parts of the
(somatic hypermutation) have better prognoses
antibody protein (Fig. 13.15). These include 123 to 129
than CLL arising from cells before the mutation
variable (VH) regions (38 to 46 functional gene seg-
process. Variable regions are sequenced as a clin-
ments) and 9 joining (JH) regions (6 functional), one
ical test in CLL. More than 2% sequence diver-
of which will connect one variable region with a con-
gence from an “unmutated” reference sequence is
stant (CH) region of the antibody or receptor. There are
considered “mutated,” with a better prognosis than
11 constant regions (9 functional). The immunoglobulin
“unmutated” variable regions.
heavy-chain gene also contains 27 diversity (DH) regions
(23 functional), one of which will connect the variable
and joining regions. The V segments are each preceded After the gene is transcribed, one of the constant regions
by a leader region (L). The leader region codes for a is joined to the final messenger RNA by splicing or,
short sequence of amino acids on the amino terminus alternatively, by a secondary recombination event (class
of the protein that marks the antibody for secretion or switching). The maintenance of the constant regions in
membrane insertion. the DNA allows for antibody-type switching during the
As B lymphocytes mature, selected gene segments are immune response.
joined together so that the rearranged gene contains only
one of each VH, DH, and JH segment (see Fig. 13.15). Ini-
tially, one DH and one JH segment are joined together. The Advanced Concepts
DNA between the two segments is looped out and lost.
The DH–JH rearrangement occurs in both alleles of the The constant region determines the isotype of the
heavy-chain gene locus on both chromosomes. Then in antibody IgM, IgD, IgG, IgE, or IgA. Each cell can
only one allele, a VH segment is chosen and joined to the make only one heavy-chain protein, although the
DH segment. The completion of the gene-rearrangement isotype of the heavy chain may change. A mature
process on only one of the two immunoglobulin
V1–70 V1–63 V1–50 V9–49 V5–48 V1–47 V7–46
V5–45 V1–44 V7–4 V1–42 VVII41–1 V1–41 V1–40 V1–38 V5–37 V1–36
V7–36 V2–34 V3–21 V1–20 V3–19
V2–18 V3–17 V3–16 V4–3 V3–2 V3–1 J1 C1
J2 C2 J3 C3 J4 C4 J5 C5 J6 C6 J7 C7
FIGURE 13.16 Immunoglobulin kappa and lambda light-chain loci consist of gene segments for variable (V), joining (J), and
constant (C) regions. The variable regions are classified into sequence-related families (V1 to Vn). Each member of the family is
given a number; for example, V7-4 is the fourth member of the V7 sequence family. Some of the gene segments are nonfunctional
(open boxes). Recombination sites (triangles) are juxtaposed to each gene segment. Arrows denote primer-binding sites for PCR
clonality testing.
V3–15 V3–11 V2–10 V1–9 V1–8 V3–7

B cell will initially produce IgD and some mem-
brane IgM that will migrate to the cell surface to
act as the antigen receptor. Upon antigen stimula- V1–6 V1–5 V2–4 V7–3 V5–2
tion, the B cell will differentiate into a plasma cell
expressing large amounts of secreted IgM. Some
cells will undergo a class-switch recombination, V4–1 J1–5 IgKC KDE
placing the VDJ gene next to the genes encoding
the IgG, IgE, or IgA constant regions. The B cells
will express a different isotype during the sec- FIGURE 13.17 Light-chain genes are rearranged in a
ondary response. Most commonly, IgM (primary kappa-before-lambda order. The rearrangement of the kappa-
response) gives way to IgG (secondary response). deleting element (KDE) eliminates the kappa locus before
Production of IgE or IgA instead of IgG can also lambda gene rearrangement. The KDE rearrangements occur
occur. Class switching is mediated by different in virtually all Ig-lambda B cells and in one-third of Ig-kappa
B cells.
recombinase enzymes than those responsible for
VDJ recombination. chromosome 2 and the lambda locus on chromosome
22. At the kappa locus, there is a single constant gene
segment, 5 joining (Jκ) gene segments, and at least
76 variable (Vκ) gene segments (30 to 35 functional)
Immunoglobulin Light-Chain Gene
belonging to 7 sequence-related families.79,80 In addi-
Rearrangement in B Cells
tion, there is a kappa deleting element (KDE) located
Like the Ig heavy-chain gene on chromosome 14, the 24 kbp 3′ to the constant region (Fig. 13.17). This
Ig light-chain genes consist of a series of gene segments element determines deletion of the Igκ constant region
in the germline configuration (Fig. 13.16). Two separate in cells producing Ig lambda light chains. The Ig lambda
genes code for the Ig light chains, the kappa locus on gene locus consists of 52 variable (Vλ) gene segments
(29 to 33 functional) from 10 Vλ families and 7 Jλ and δ chains are encoded on chromosome 14 (the δ gene
(4 to 5 functional) and 7 to 11 Cλ gene segments (4 to 5 is located inside of the α gene), and γ and β are located
functional) occupying 1,140 kb of DNA.81 on chromosome 7. The four chains form pairs, making
Immunoglobulin light-chain gene rearrangement is two types of receptors, αβ and γδ. T-cell receptor genes
similar to that described for the immunoglobulin heavy- have fewer variable gene segments than the immuno-
chain gene. Selected gene segments are joined together, globulin genes, and the genes for the γ and α chains
with loss of the intervening DNA and possible insertion have no diversity regions (Table 13.6). The V regions
of nucleotides at the junction. The kappa locus rear- of the receptor chains undergo gene rearrangement by
ranges first and then the lambda locus, if necessary. If intrachromosomal recombination as described for the
the lambda locus rearranges, the kappa locus undergoes immunoglobulin genes (Fig. 13.18).
a secondary recombination through the KDE so that the
cell does not produce both types of light chains.
During differentiation of the B cells from precursor
stem cells, rearrangement, recombination, and mutation
Advanced Concepts
of the immunoglobulin V, D, and J regions ultimately
Antibody diversity is ensured by three separate
results in functional VJ (light-chain) and VDJ (heavy-
events during and after the gene-rearrangement
chain) genes.
process. The first is the selection of gene seg-
ments. The second is the imprecise joining of the
T-Cell Receptor Gene Rearrangement
segments together with the addition of nucleo-
The T-cell receptor is composed of two of four chains, tide bases at the junction.82 Third, after the gene-
α, β, γ, and δ, with characteristic structures resembling rearrangement process has finished and the B cell
immunoglobulin V, J, and C regions (Fig. 13.18). The α
D J
V V 1 2 1 2 C V J J2 C
V
1 2 VN D1 J1 C D2 J2 C2 V
FIGURE 13.18 General structure of the T-cell
receptor genes. The gene for the delta T-cell
V receptor chain is contained in the alpha locus
1 2 3 4 5 6 7 8 VA 9 10 B 11 JP1 J J1 C1 JP2 J2 C2 (top). The beta and gamma chains are located at
separate loci.
TABLE 13.6 T-Cell Receptor Gene Segments
T-Cell Receptor Variable Gene Diversity Gene Joining Gene Constant Gene
Chain Segments* Segments Segments* Segments
α 54/45 0 60/50 1
β 67/47 2 14/13 2
δ 3 3 4 2
γ 14/6 0 5 2
*Total gene segments/functional gene segments

λδ receptors can represent a predominant population in

has encountered antigen, somatic hypermutation certain tissues, such as the intestinal tract.
occurs in the variable regions of the rearranged
heavy- and light-chain genes. In addition to the Detection of Clonality
deaminase, the mutation process requires the action
Gene rearrangements occur independently in each lym-
of repair systems that further substitute alternate
phocyte so that a normal population of lymphocytes is
nucleotides in the sequence, resulting in different
polyclonal (polytypic) with respect to their rearranged
amino acid substitutions in the antibody protein.
immunoglobulin or T-cell receptor genes. Overrepre-
Changing of the variable-region sequences under-
sentation of a single rearrangement in a specimen cell
lies the process of affinity maturation. As the
population can be an indication and characteristic of a
B cells replicate, those producing antibodies with
lymphoma or leukemia. When over 1% of cells make
greater affinity to the antigen are favored, generat-
the same gene rearrangement, the cell population is
ing subclones of cells that may replace the origi-
referred to as monoclonal (monotypic) with respect to
nal reactive clone. Over the course of an infection,
the rearranged genes.
therefore, antibodies with increased affinity are
produced.
Advanced Concepts
It is important to distinguish a large monotypic
Advanced Concepts population of tumor cells from a reactive clone or
oligoclone or reactive clone of cells responding to
The T-cell receptor δ gene is flanked by TCR an antigen. Oligoclones are not only smaller but
δ–deleting elements. Recombination between transient in nature, so they should not be consis-
these elements or between Vα and Jα results in tently present in serial analyses.
deletion of the TCR δ gene.
Molecular Analysis of Immunoglobulin

Heavy-Chain Gene Clonality
Rearrangement of the T-cell receptor chains proceeds in
a similar manner as in the immunoglobulin genes. The Clonality can be detected by protein and nucleic acid
V, (D), and J segments are joined together with the addi- analyses. The first nucleic acid tests for gene rearrange-
tion or deletion (trimming) of nucleotides at the junc- ments were performed by Southern blot.83 With regard to
tions between the gene segments (Fig. 13.19). the immunoglobulin heavy-chain gene, restriction sites
The extracellular domains of the T-cell receptor were mapped in the germline configuration. BamH1,
dimers are held in conformation by interchain disulfide EcoR1, and HindIII were restriction enzymes commonly
bridges between cysteine residues in the T-cell receptor used for this procedure (Fig. 13.20). When the germline
peptides. The T-cell receptor chains also have a hydro- sequence is rearranged, the restriction sites are moved,
phobic transmembrane region and a short cytoplasmic created, or deleted, resulting in a unique restriction
region. Although most cells express the αβ receptor, pattern for every gene rearrangement.
V1 2 3 4 5 6 7 8 VA 9 10 B 11 JP1 JP J1 C1 JP2 J2 C2
FIGURE 13.19 T-cell receptor gamma

gene rearrangement occurs through
selection of variable (V) and joining (J) V1 2 3 4 5 6 7 8 VA 9 J2 C2
segments.
11 kb
Hind III Hind III
18 kb
BamHI Bam HI
L VH1 L VHN DH JH C
18 kb
EcoRI EcoRI
Labeled probe
FIGURE 13.20 Restriction map of the germline immuno-

globulin heavy-chain gene. The fragments indicated by the
arrows will be detectable with the probe shown at the bottom.
Gene rearrangement will affect the placement of the restriction
sites such that fragments of different sizes will be generated
from a rearranged gene.
For the Southern blot, DNA cut with the restriction FIGURE 13.21 Colorimetric results from an immunoglobu-
enzymes is transferred to a nitrocellulose membrane and lin heavy-chain gene rearrangement test by Southern blot with
probed to the joining region of the gene. Normal results colorimetric detection. Lane 1, molecular-weight markers.
should reveal the expected fragments generated from Lanes 2, 6, and 8 show the normal 18-kbp, 18-kbp, and 11-kbp
the germline DNA, in the example, an 18-kbp EcoR1 bands expected from the germline gene in a normal specimen
fragment, an 18-kbp BamH1 fragment, and an 11-kbp cut with EcoR1, BamH1, and HindIII, respectively. Lanes 3, 6,
HindIII fragment. The normal fragments are visible for and 9 show patient DNA with a monoclonal cell population.
Lanes 4, 7, and 10 show a patient with no detectable
two reasons. First, a normal patient specimen contains
monoclonality.
cells other than lymphocytes that do not undergo gene
rearrangement. The second reason for the presence of
the germline bands is that only one chromosome in a
lymphocyte undergoes gene rearrangement, leaving Advanced Concepts
the homologous chromosome in the germline state. If
the first rearrangement fails or is unproductive, then the Three enzymes are used for this assay to avoid
second chromosome will rearrange. This occurs in fewer false-positive results due to cross-hybridization
than 10% of lymphocytes for the heavy-chain gene. artifacts. The chance of true rearranged bands
In a normal specimen, there will be millions of immu- being identical to cross-hybridization patterns
noglobulin gene rearrangements, but no one rearrange- for all three enzymes is negligible. In addition,
ment is present in high enough amounts to be visible cross-hybridization patterns are constant with the
as nongermline bands on the membrane or autoradio- same hybridization conditions, in contrast to true
gram; thus, only the germline bands will be visible. If, monoclonal gene-rearrangement bands that differ
however, 2% to 5% of the cells in the specimen consist for each monoclonal population.
of a clone of cells, all with the same gene rearrangement,
that clone will be detected by the presence of additional
bands different from the germline bands (Fig. 13.21). Analysis of clonality by Southern blot affords the advan-
Interpretation of the results is positive if extra bands tage of detecting all gene rearrangements, including
are present and negative if only the germline bands are incomplete rearrangements involving only the D and J
present. regions of the gene. The Southern blot method is limited
by the requirement for at least 20 to 30 μg of high- L V DJ C

quality DNA from the specimen. This is not always
available, either because the specimen is limiting with
respect to cell number or because it is of limited quality,
as in paraffin-embedded specimens. Moreover, artifacts
FR1 CDR1 FR2 CDR2 FR3
such as cross-hybridizations may complicate the inter-
pretation of Southern blot results.
PCR is the most frequent approach for the detec- Amplification
tion of gene rearrangements. For this method, forward
primers complementary to the variable region and
reverse primers to the joining or constant regions of the
B-cell–rearranged genes are used.84 The resulting ampl-
icons are resolved by agarose, polyacrylamide, or capil-
lary electrophoresis. A normal cell population will yield Amplification products
amplicons consisting of fragments of different lengths,
yielding a cloud or smear of bands, or series of peaks. A
monoclonal cell population will yield a predominant or
single band or peak, the presence of which is interpreted
as a positive result. A limitation of gene rearrangement
studies by PCR is the inability to amplify all possible
gene rearrangements as primer binding sites are lost
during recombination and somatic mutation.
A limitation to the detection of clonality by PCR is
the loss of the FR3 primer-binding site due to the gene- FIGURE 13.22 Immunoglobulin heavy-chain gene rear-
rearrangement process, which may destroy or remove rangement by PCR with amplification from the variable region.
the sequences bound by the FR3 primer. To address this Forward primers complementary to the variable region and
issue, some laboratories use additional forward primers reverse primers complementary to the joining region are used
that bind to framework 2 (FR2) and framework 1 (FR1). to amplify the diversity region (top). In a polyclonal specimen,
Other methods utilize primers to the leader region in amplification products in a range of sizes will result. These
addition to the FR primers.84,85 These primers increase products produce a dispersed pattern on an ethidium bromide–
the number of gene rearrangements that can be ampli- stained agarose gel (lanes 4, 8, and 11). If at least 1% of the
fied and therefore detected by this assay. Deletion of the sample is representative of a monoclonal gene rearrangement,
IgH locus can occur in some lymphomas, precluding that product will be amplified preferentially and revealed as a
sharp band by gel electrophoresis (lanes 3, 6, 9, and 10).
amplification with any primers.86 If the gene rearrange-
ment cannot be amplified, cytogenetics may be used
to assess clonality at the immunoglobulin heavy-chain
gene locus.87 more stable in sequence. Standard methods for clonal-
For immunoglobulin heavy-chain gene rearrange- ity utilize a forward consensus primer to the innermost
ment, different approaches to the variable-region primer- framework region (FR3) and the reverse primer com-
binding sites have been taken (Fig. 13.22). The immu- plementary to the joining region. The consensus primer
noglobulin heavy-chain gene-variable region is divided has sequences that match the most frequently occurring
into two types of domains. The complementarity- sequences in the FR3 region and may not be identical
determining regions (CDRs) code for the amino acids to any one sequence. Enough nucleotides will hydro-
that will contact the antigen. The CDRs, therefore, are gen bond, however, to match most rearranged variable
the most variable, or unstable, in sequence. The frame- regions. Primers directed at the diversity region are
work regions (FRs) code for the amino acids that have useful for amplification of the germline configuration
more of a structural role in the antibody protein and are of the immunoglobulin heavy-chain genes as well as
L V DJ C Immunoglobulin Light-Chain
Gene Rearrangements
The immunoglobulin light-chain genes are also targets
for clonality detection. In addition to protein analy-
FR1 CDR1 FR2 CDR2 FR3 sis, Southern blot, PCR and RT-PCR have been used
to detect light-chain gene clonality.91–93 Targeting the
DH7 immunoglobulin light-chain genes is especially useful
FIGURE 13.23 Immunoglobulin heavy-chain gene rear- for tumors arising from terminally differentiated B cells
rangement by PCR with amplification from the diversity (plasma cells) that have undergone extensive somatic
region. Forward primers complementary to the diversity region hypermutation at the heavy-chain gene locus. In these
and reverse primers complementary to the joining region yield tumors, the rearranged heavy-chain genes are frequently
a polyclonal pattern in normal samples. The DH7 primer will unamplifiable, yielding false-negative results because
yield a specific 350-bp product from the unrearranged (germ- of accumulation of base changes in the variable-region
line) gene due to the short distance between the DH7 gene primer-binding sites. Most of these tumors are amplifi-
segment and the joining-region primer. able, however, at the light-chain genes.94
Gene rearrangements in the kappa light-chain locus
are amplified using primers complementary to the
sequence families of the Vκ region or to the intron
DH–JH rearrangements.88 Primers complementary to the
between the Jκ regions and Cκ. Opposing primers are
seven family-specific sequences of the diversity region
complementary to Cκ and to one or more of the five
and a primer to the 5′-most joining region (JH1) are
joining regions. Alternately, the KDE can be used for a
used to amplify the DH–JH junction. The primer comple-
primer-binding site (Fig. 13.24). Using the KDE allows
mentary to the DH7-27 segment, the sequences closest
detection of lambda-expressing cells that have deleted
to the joining region, will yield a defined product from
Jκ or Cκ.92
the Ig heavy-chain gene in the germline configuration
Immunoglobulin heavy-chain gene rearrangement
(Fig. 13.23). Diversity-region primers are also useful for
precedes Ig light-chain gene rearrangement during early
targeting incomplete rearrangements where the variable-
B-cell differentiation in the bone marrow. Following
region primer-binding sites are lost.89,90
successful IGH recombination, the Ig light-chain loci
Another approach to the detection of B-cell clonality
then rearrange, first at the Ig kappa locus. If the kappa
is to make patient-specific primers. For this method, a
rearrangement is nonfunctional, the Ig lambda locus
consensus primer is used to amplify the rearranged gene
rearranges. With functional heavy- and light-chain gene
from a positive specimen. The amplification product is
rearrangements, the cell will develop into a mature naïve
then purified from the gel or from the PCR reaction mix
B cell. Therefore, detection of clonality at the Igλ locus
and sequenced. Primers exactly matching the variable-
on chromosome 22 is also useful for confirming or mon-
region sequence of that specimen are then manufactured
itoring diagnosis of B-cell leukemias and lymphomas.
for use on subsequent samples. The advantage of this
Forward primers complementary to Vλ gene segments
method is that it is more sensitive because fewer or
and reverse primers to the Jλ and Cλ gene segments are
none of the gene rearrangements from normal cells are
frequently used for these assays (Fig. 13.25).
amplified. Furthermore, the tumor load can be measured
quantitatively by real-time PCR (RT-PCR). The disad-
T-Cell Receptor Gene Rearrangements
vantage of this approach is that the method is more time-
consuming to perform, and the primers used are patient Like B-cell clonality and B-cell malignancies, clonal-
specific. Moreover, in a patient with a chronic condition, ity of T-cell receptor gene rearrangements may demon-
for whom monitoring is likely to be done, tumor cells strate that a high white cell count is of T-cell origin or
may undergo further mutation in the variable region the presence of a clonal T-cell infiltrate accompany-
and inactivate the specific primer binding, requiring the ing another malignancy. Clonality may also be used to
manufacture of new primers. monitor treatment efficacy. Clonal T-cell receptor gene
VK JK Intron CK
VK2 VK4 VK5 VK3 VK6 VK7
VK JK Intron CK KDE
FIGURE 13.24 The immunoglobulin light-

chain kappa locus can be amplified by standard
PCR procedures from the variable region, the VK KDE VK JK Intron KDE
intronic region, or the kappa-deleting element.
J1 C 3
V J Intron C
V1
FIGURE 13.25 The immunoglobulin light-chain lambda

locus can be amplified by standard PCR procedures from the
variable region to the joining region or the constant region.
Reverse transcriptase PCR is used with the constant-region
primers to eliminate the intron between the constant and
joining regions.
rearrangements are detected in a manner similar to the

immunoglobulin gene rearrangements by Southern blot,
PCR, and sequencing.95 For Southern blot studies, probes
complementary to the variable, joining, and diversity
regions of the T-cell receptor genes are used to detect
monoclonal populations. Just as with immunoglobulin
gene rearrangements, no one gene rearrangement should FIGURE 13.26 T-cell receptor gene rearrangements detected
be visible in a normal specimen. The presence of a large by Southern blot using a probe mixture of sequences comple-
monoclonal population is revealed by the bands detect- mentary to Jβ1 and Jβ2. Lane 1, molecular-weight markers.
able in addition to the germline bands seen in the normal Lanes 2, 5, and 8, normal control cut with EcoR1, BamH1, and
HindIII, respectively. Lanes 3, 6, and 9, a negative specimen
control (Fig. 13.26).
cut with EcoR1, BamH1, and HindIII, respectively.
The T-cell receptor gene rearrangement assays done
Lanes 4, 7, and 10, positive specimen cut with EcoR1, BamH1,
by PCR most often target the TCRγ gene. Assays are and HindIII, respectively. Note the additional bands in the
also performed on the TCRβ and TCRδ genes. Detec- positive specimen lanes.
tion of gene rearrangements in TCRα is difficult due to
the 85-kb length of the Jα gene segments. TCRα gene
rearrangements may be inferred from TCRδ gene dele-
tions.96 Primers are designed complementary to the
rearranged gene segments (Fig. 13.27). Multiple primer heteroduplex analysis may be used to improve the reso-
sets are often used to ensure detection of the maximum lution of the polyclonal and monoclonal patterns.
potential gene rearrangement.97 PCR will yield a product In 2003, a primer set was designed by the European
consistent with a single-gene rearrangement in a positive Commission Biomed-2 group to amplify an increased
sample, whereas a normal sample will yield a polyclonal number of possible immunoglobulin heavy-chain gene
pattern (Fig. 13.28). Due to the limited range of lengths rearrangements.84 This set comprises 107 primers multi-
of the population of rearranged T-cell receptor genes, plexed in 18 PCR reactions. In addition to immunoglob-
ulin heavy-chain, T-cell receptor gene rearrangements,
J these primers are directed at two translocations, t(14;18)
and t(11;14) (see following discussion).
V D1 D2 Advanced Concepts

D3 J
T-cell receptors do not undergo somatic hypermu-
tation when a given T cell is exposed to antigen.
Therefore, the specificity of a given T-cell clone
V D2
J1 J2
maintains a common antigen specificity, which is
the clonotype of that population. Massive parallel
sequencing of T-cell receptors and alignment of
the data to the germline gene sequences can reveal
V1 V9 V10 V11 a rearrangement. By taking a tally of the reads, a
full picture of the T-cell receptor clonotype can be
FIGURE 13.27 T-cell receptor gene rearrangements detected
by PCR use primers to the variable, diversity, and joining
generated.98
regions of the rearranged genes.
Banding Patterns
Interpretation of clonality by PCR depends on gel
banding patterns. False-negative results may arise from
primers that do not match the gene rearrangement in the
tumor. On the other hand, artifactual single bands will
produce false-positive results. Patterns of multiple single
bands may arise from specimens with low cell numbers,
such as cerebrospinal fluid or paraffin sections. In this
case, usually more than one or two single bands will
occur. The bands may not indicate monoclonality but,
rather, that only a few cells are present. These cells may
or may not be malignant. False single-band patterns may
also occur within polyclonal smears detected on non-
denaturing polyacrylamide gels (PAGEs). Heteroduplex
FIGURE 13.28 T-cell receptor gene rearrangement by PCR analysis is recommended if amplicons are resolved by
and heteroduplex analysis. Bands were separated by polyacryl- nondenaturing PAGEs.
amide gel electrophoresis and stained with ethidium bromide. Similar problems occur with resolution by capil-
Lanes 2 and 4 show a polyclonal pattern. A positive result lary electrophoresis where peak heights and widths are
(monoclonal pattern) is shown in lane 3. Lane 1, molecular- compared to distinguish wide polyclonal peaks from
weight marker; lane 5 reagent blank. narrow monoclonal spikes.99 Pretreatments such as
eosin staining may yield false single bands by capillary IgH

electrophoresis.100 Chromosome 14
X
Mutations in Hematological Malignancies BCL2
Chromosome 18
Lymphoid Malignancies
Immunoglobulin and T-cell receptor gene rearrangements
are normal and take place regardless of the presence of BCL2 IgH
malignancy. The unique nature of the gene-rearrangement t(14;18) translocated chromosome
system with cell-specific antibody and antibody receptor FIGURE 13.29 The t(14;18) translocation moves the BCL2
formation is exploited in finding abnormal cell popula- gene intact to the long arm of chromosome 14 next to the
tions clonally derived from these cell-specific features. joining region of the immunoglobulin heavy-chain gene (IgH).
Targeting these gene rearrangements, however, can only The translocation breakpoints on chromosome 18 are 3 + to the
be applied to tumors arising from lymphocytes. Other BCL2 gene (arrow indicating the direction of transcription).
types of malignancies, such as myeloid tumors, cannot
be analyzed using these targets.
Often, the development of the cancer state results in
recognition sites for the gene-rearrangement recombi-
DNA anomalies, such as translocations or other types of
nase enzymes on chromosome 18. As a result, an abnor-
mutations. These can be applied to the clinical discov-
mal interchromosomal exchange occurs instead of the
ery and monitoring of tumors. These abnormal genetic
normal intrachromosomal exchange events described
events aid in the diagnosis of specific types of hemato-
in the previous section (Fig. 13.29). There are several
logical tumors, such as the translocations that are highly
breakpoints in the chromosome 18 region 3′ to the BCL2
associated with chronic myelogenous leukemia, promy-
gene, most of which occur in the major breakpoint region
elocytic leukemia, or follicular lymphoma. Transloca-
(MBR). About 10% to 20% of the breakpoints fall into
tions are the exchange of DNA between chromosomes.
a cluster closer to the BCL2 gene, thousands of bases
If the translocation disrupts the activity or expression
from the MBR, in the minor cluster region (MCR). An
of oncogenes or tumor-suppressor genes, a cancer phe-
intermediate cluster region (ICR) and other breakpoints
notype can occur. Several translocations are frequently
outside of MBR and MCR have also been reported.101
found in certain types of tumors.
Breakpoints in this and other translocations are associ-
t(14;18)(q32;q21) ated with double-helix disruption or fragile sites in the
The reciprocal translocation between the long arms of DNA.102
chromosomes 14 and 18 moves and dysregulates the Molecular detection of the t(14;18) translocation is
BCL2 gene located on chromosome 18q21.3. BCL2 performed by a variety of methods. Southern blot with a
(B-cell leukemia and lymphoma 2 or B-cell CLL/lym- probe to the MBR region of chromosome 18 will reveal
phoma 2) is an oncogene. The gene product of BCL2 is a the translocation by the presence of bands different
member of a group of related proteins that control apop- from those expected from a normal chromosome 18
tosis (cell death initiated by internal cellular signals). (Fig. 13.30). The translocation is easily and rapidly
The Bcl2 protein inhibits apoptosis in B lymphocytes, detected by PCR or qPCR. Forward primers to chromo-
that is, enhances survival of cells that normally would some 18 and reverse primers complementary to the Ig
die. Survival of genetically damaged cells may contrib- heavy-chain joining region will yield a product only if
ute to the development of tumors. One of the most fre- the two chromosomes have been joined by the transloca-
quent hematological malignancies, follicular lymphoma, tion. The amplicons are visualized by gel electrophore-
is associated with the t(14;18) translocation. sis, as shown in Figure 13.31, or with a qPCR probe. The
The translocation partner of chromosome 18 is chro- t(14;18) translocation can be used to quantify tumor load
mosome 14 in the vicinity of the immunoglobulin heavy- by qPCR. Several methods are available for this analy-
chain gene. The translocation may occur through cryptic sis, and although laboratory methods differ, the results
cells, the standard curve generates the formula

y = 1.5631[Ln(x)] + 42.396, where y is the threshold
cycle number, and x is the number of translocated cells
(50 ng of DNA was used per sample). Using this formula,
if a given sample crosses the fluorescence threshold at
y = 36 cycles (average of duplicate measurements), the
number of cells (x) is approximately 60 cells. Assum-
ing that 50 ng of DNA represents 7,500 cells (1 ng of
DNA = approximately 150 cells), 60/7,500 = 0.008.
0.008 × 100 = 0.8% translocated cells in the specimen.
For the t(14;18) translocation, sensitivities of 0.0025%
have been reported, with a linear range of 0.01%
to 10%.104
The main limitation of any PCR procedure targeting
the t(14;18) translocation is the inability of the primers
to detect all of the possible breakpoints on chromo-
some 18. Primer pairs, sets of primer pairs, and novel
probe methods have been designed to address this
problem.86,105 Test reports should include an estimation
of false-negative results expected due to breakpoints that
remove the binding sites for the primers used. Unless a
FIGURE 13.30 Analysis of the t(14;18) gene translocation translocation has been previously observed by a given
by Southern blot with chemiluminescent detection. Lanes 3, 6, PCR method, a negative result should acknowledge that
and 9 are the normal control cut with EcoR1, BamH1, and
a translocation may be present but undetectable with the
HindIII, respectively. Lanes 1, 4, and 7 show results from a
primers used.
normal patient specimen. Lanes 2, 5, and 8 are results from a
specimen positive for the t(14;18) translocation. Note the extra
bands in the positive specimen. t(11;14)(q13;q32)
The t(11;14)(q13;q32) translocation joins the immuno-
globulin heavy-chain gene region on chromosome 14
were found to be reasonably consistent in comparison with part of the long arm of chromosome 11. The cyclin
testing.103 Most methods include a standard curve for D1 (CCND1) gene, also called the parathyroid adeno-
regression analysis of the test sample measurements matosis 1 gene (PRAD1) or BCL1, on chromosome 11 is
(Fig. 13.32). Alternatively, internal controls, such as attached to the long arm of chromosome 14 in the intron
known amounts of plasmid DNA, may be added to the between the immunoglobulin heavy-chain gene joining
test specimens. In this method, the amount of translocated and constant regions. The translocation increases expres-
cells (or translocated chromosomes) is determined rela- sion of CCND1, resulting in passage of the cell cycle
tive to the internal control. There are several approaches from the G1 to the S phase of the cell cycle. This trans-
to reporting the final results. Most frequently, the results location is found primarily in mantle cell lymphoma
are reported as the percentage of translocated cells in the (MCL) but may also be present in chronic lymphocytic
specimen tested. For instance, the raw number of trans- leukemia, B-prolymphocytic leukemia, plasma cell leu-
located cells is determined using the standard curve of kemia, multiple myeloma, and splenic lymphoma. The
threshold cycles versus the known cell count. The raw t(11;14) translocation is thought to be a definite charac-
number of cells is then divided by the number of total teristic of MCL; however, only 50% to 70% of MCLs
cells represented in the PCR reaction. have a detectable t(11;14).
For example, for a dilution series ranging from 50 Mantle cell lymphoma without the t(11;14) translo-
to 10,000 translocated cells in 1 million untranslocated cation is likely to show expression of the transcription
JH primers
BCL2 gene IgH
MBR primers MCR primers
FIGURE 13.31 Analysis of the t(14;18) gene translocation by PCR with agarose gel electrophoresis. Several primers are used for
detection of the translocated chromosome (top). Because the forward primers are on chromosome 18 and the reverse primers are
on chromosome 14, a PCR product will be generated only from the t(14;18) translocated chromosome (bottom). M, molecu-
lar-weight marker; +, positive specimen; -, negative specimen; P, positive control; S, sensitivity control; N, negative control.
40
35
factor SOX11. SOX11 therefore represents an important
Cycle
30 performed by IHC or western blot.106

Methods for detection of t(11;14) are similar to those
25 used for t(14;18) translocation described previously.
10 100 1,000 10,000
Cells
Southern blot methods have been replaced mostly with
PCR and RT-PCR,107 but due to the variation in break-
points, primers can miss some translocations. In these
cases, FISH or flow-cytometry analysis may be prefer-
able.108 Of the breakpoints on chromosome 11, 80% are
in the major translocation cluster 5′ to the CCND1 gene
(Fig. 13.33). The rest are dispersed in other areas 5′ or
3′ of the gene. PCR analysis detects 40% to 60% of the
FIGURE 13.32 The t(14;18) translocation analysis by qPCR.
A standard curve is established using cultured cells with the translocation breakpoints.109 A PCR product will result
t(14;18) translocation counted and mixed at different propor- only if the translocation has occurred (and the primer
tions with cultured cells that do not have the translocation. The binding sites are maintained). The PCR product can be
illustration represents DNA isolated from each mixture of cells detected by qPCR fluorescence or by gel or capillary
analyzed by qPCR with a TaqMan probe. electrophoresis.
Chromosome 11 Chromosomes:
MTC MTC2 MTC3 CCND1 14 8 t(8,14) translocation
Chromosome 14
IgHC J D V
IgHC J CCND1
t(11;14)
FIGURE 13.33 The t(11;14) breakpoints in chromosome 11

(top) and chromosome 14 (center) are indicated by the vertical
arrows. Primers are complementary to the joining region of the
immunoglobulin heavy-chain gene and the CCND1 gene
(bottom).
FIGURE 13.34 The t(8:14) breakpoint detected by CISH

Advanced Concepts (Invitrogen). Two probes labeled with biotin or digoxigenin are
complementary to sequences flanking the chromosome 14
A variety of molecular events can result in the over- breakpoint. In the absence of the translocation (left), the probes
will appear next to one another in the nucleus. The transloca-
expression of c-myc. The DNA virus Epstein–Barr
tion (right) will move one probe to chromosome 14, leaving
virus (EBV) is associated with Burkitt lymphoma. the other behind on chromosome 8, resulting in separation of
EBV induces cell proliferation and, thus, provides the signals in the nucleus.
more opportunity for translocation events. Certain
types of another DNA virus, human papillomavi- promoter and regulatory region and moved into the
rus, inserted into the vicinity of c-myc cause over- switch recombination region of the immunoglobulin
expression of the gene.110 heavy-chain gene on chromosome 14. In the t(2;8) and
the t(8;22) translocations, the chromosome 8 break-
points are 3′ to the gene, and c-myc is moved into the
t(8;14)(q24;q11) immunoglobulin kappa or lambda locus, respectively.
The avian myelocytomatosis viral oncogene homolog Translocations of c-myc into the T-cell receptor alpha
(c-myc) gene on chromosome 8 is one member of a gene also occur.111
gene family including n-myc and l-myc. The c-myc gene In the laboratory, c-myc translocations were com-
codes for a helix-loop-helix/leucine zipper transcription monly detected by Southern blot analysis with a
factor that binds to another protein, Max, and activates probe complementary to exon 3 of the c-myc gene;
transcription of other genes. The t(8;14) is associated for instance, a 32P- or digoxigenin-labeled 1.4-kb ClaI-
with Burkitt lymphoma; in addition, translocations at EcoRI restriction fragment. Interphase FISH and CISH
(2;8) and t(8;22) are found in about 10% of Burkitt are now used to detect the t(8;14) translocation with
lymphomas. separate probes to chromosomes 8 and 14 or with chro-
In the t(8;14) translocation, the breakpoints on chromosome 14 probe pairs (Fig. 13.34). Amplification of
mosome 8 are spread over a 190-kbp region 5′ to and the gene can be detected by FISH using a 120-kb c-myc
within the c-myc gene. As a result of the t(8;14) trans- (8q24.12-q24.13) fluorescent probe. Myc protein expres-
location, the c-myc gene is separated from its normal sion is measured by IHC.112
detection of translocations by PCR is used for t(9;22);

Myeloid Malignancies
that is, forward primers are designed to hybridize to the
t(9;22)(q34;q11) BCR gene on chromosome 22 and reverse primers to
The t(9;22) translocation is a reciprocal exchange chromosome 9 in the c-abl gene (Fig. 13.37). A product
between the long arms of chromosomes 9 and 22. The will result only if the two genes are joined by the trans-
translocation generates the Philadelphia chromosome location. Due to the length of the introns that separate
(Ph1), which is present in 95% of cases of chronic the primer-binding sites, nested RT-PCR was initially
myelogenous leukemia (CML), 25% to 30% of adult utilized.113 To minimize the risk of false-positive results,
acute lymphoblastic leukemia (ALL), and 2% to 10% this method may require confirmatory testing, especially
of pediatric ALL. The breakpoints of the t(9;22) transfor adult ALL.114 For optimal performance, primers
location occur within two genes, the breakpoint cluster capable of detecting both major and minor breakpoints
region (BCR) gene on chromosome 22 and the cellu- in the BCR gene are utilized. An internal RNA integrity
lar counterpart of the Abelson leukemia virus tyrosine control (or amplification control) is included for each
kinase (c-abl) on chromosome 9 (Fig. 13.35). The result sample to avoid false-negative results from poor RNA
of the translocation is a chimeric or fusion gene with the quality or inadequate cDNA synthesis. Transcripts from
head of the BCR gene and the tail of the c-abl gene. Both the abl or BCR genes are used most frequently for the
genes are tyrosine kinases; that is, they phosphorylate RNA integrity control for the t(9;22) translocation, but
other genes at tyrosine residues. The fusion gene is also other unique genes with constitutive expression have
a kinase but has aberrant kinase activity. There are two been used. Target amplicons are detected by agarose
major forms of the BCR/abl fusion gene, joining either gel electrophoresis with ethidium bromide staining
exon 13 or 14 (b2 or b3) of the BCR gene to c-abl exon (Fig. 13.38) or by capillary gel electrophoresis. Fluo-
2 (a2; Figure 13.36). The b2a2 or b3a2 fusion genes rescent dye–labeled primers are required for the latter
code for a 210-kd protein, p210. A third form of the detection method.
fusion gene joins exon 1 of BCR with exon a2 of c-abl, Quantitative PCR provides an estimation of treatment
resulting in the expression of an e1a2 transcript, which response, especially with targeted therapies for CML
codes for a p190 protein. Another, less common, fusion and ALL. Although cytogenetic methods, especially
junction occurs at exon 19 of the BCR gene (c3). The FISH and standard RT-PCR, are most practical for diag-
c3a2 transcript encodes a p230 protein. All of the fusion nosis and in the early stages of treatment, quantitative
proteins have been observed in CML; however, p190 PCR provides a valuable estimation of tumor load over
occurs mostly in ALL. The common rationale for the the course of treatment.115–117
BCR gene on chromosome 22

m-BCR M-BCR
minor breakpoint major breakpoint
e1 b1 b2 b3
c-abl gene on chromosome 9

1B 1A a2 3 4 5 6 7 8 9 10 11
FIGURE 13.35 The t(9;22) translocation begins with breakage of chromosomes 9 and 22 in introns of the BCR and c-abl genes
(arrows).
e1 b1 b2 b3 a2 3 4 5 6 7 8 9 10 11
e1… …b3 a2… …11

Fusion mRNA AAAAA
(8.5 kb)
Fusion protein p210bcr-abl
e1 b1 b2 b3 a2 3 4 5 6 7 8 9 10 11
e1 a2… …11
Fusion mRNA
(7 kb)
Fusion protein p190bcr-abl
FIGURE 13.36 p210 and p190 are the two main fusion proteins produced by the t(9;22) translocation. They differ in the amount
of the BCR gene that is attached to the c-abl gene.
e1 a2 3 4 5 6 7 8 9 10 11
e1 b1 b2 b3 a2 3 4 5 6 7 8 9 10 11
1B 1A a2 3 4 5 6 7 8 9 10 11
FIGURE 13.37 Location of primers (horizontal arrows) for PCR analysis of the minor breakpoint region (top) and the major
breakpoint region (middle). The c-abl gene itself may be used as an amplification control (bottom). The intron and exon lengths
are not drawn to scale. Vertical arrows denote locations of breakpoints.
Because transcript levels are being measured using method are similar to those used for standard PCR. A
RT-PCR, it is important to stabilize the specimen RNA fluorescent probe provides the signal. A standard curve
on receipt, for example, by resuspending the white blood or a high positive, low positive (sensitivity) control and
cells in protective buffers. Another recommendation negative control should accompany each run. Frequently
is to collect sufficient peripheral blood for analysis to used methods reported measurements as a ratio of the
avoid false-negative results.118 The primers used for this BCR-abl transcript level to the RNA integrity control,
1 2 3 4 5 6 7 8 9 10 11 12 Therapeutic tyrosine-kinase inhibitors that target the

chimeric BCR-abl protein have changed CML from
a deadly disease to a chronic condition, treatable by
a pill a day. With the success of these agents, and to
detect resistances, efforts have been made to establish
a standardized measurement scale to provide consistent
results among laboratories.123,124 Reference standards
were established to be used to normalize measurements
from diverse laboratory methods.125 These reference
standards were protected RNA molecules or “armored
RNA.” Armored RNA is an engineered MS2 phage
protein complex encapsulating an RNA fragment of the
FIGURE 13.38 Results of a standard RT-PCR test for the BCR-abl target gene designed for use as a qPCR calibra-
t(9;22) translocation. Lane 1, molecular-weight marker; lane 2, tion standard. The protected RNA is resistant to RNase
b3a2 breakpoints; lane 3, b2a2 breakpoints; lanes 4 and 5, e1a2 digestion and shows high stability in plasma and other
breakpoints; lane 6, negative specimen; lanes 7 to 11 are RNA fluids. Sets of known concentrations of these reference
integrity (amplification) controls for specimens in lanes 2 to 6; standards are distributed to laboratories, where they are
lane 12, reagent blank. included in the laboratory measurement. The results
are sent to a test center, where the testing laboratory
usually the abl transcript, the BCR transcript, or the tran- results are adjusted to the actual numbers for the stan-
script of a housekeeping gene, such as G6PDH.115,119 For dards with a correction parameter (CP) or conversion
example, a standard curve for transcript number gener- factor (CF). The CP or CF is then applied to standard-
ates the formula y = –1.7318[Ln(x)] + 48.627, where ize the BCR-abl test results.126 Alternatively, commercial
y is the threshold cycle number, and x is the number reagent sets containing internal calibrators and con-
(or dilution) of transcripts. If quantitative PCR analysis trols may be used. These systems will generate results
of the patient specimen RNA yielded a threshold cycle on the International Scale without the requirement for
number of 39 (average of duplicate samples) for BCR- adjustments.
abl transcripts and a threshold cycle number of 30 for
the abl transcripts, then solving for x yields 300 BCR- t(15;17)(q22;q11.2-q12)
abl transcripts and 50,000 abl transcripts in the sample. The t(15;17)(q22;q11.2-q12) reciprocal translocation
Thus, (300/50,000) × 100 = 0.6%. between the long arms of chromosomes 15 and 17 results
Data are still being collected with regard to the clin- in fusion of the retinoic acid receptor alpha (RARA) gene
ical significance of the quantitative results. A three-log on chromosome 15 with the myelocytic leukemia (MYL
drop in transcript levels and a BCR-abl/abl × 100 level or PML) gene on chromosome 17. Both genes contain
below 0.05% have been proposed as indicators of good zinc finger–binding motifs and therefore bind DNA as
prognosis.120,121 transcription factors. The PML/RARA fusion is found
specifically in promyelocytic leukemia. The presence of
this translocation is also a predictor of the response to
Advanced Concepts retinoic acid therapy that is used as a treatment for this
disease. The translocation forms a fusion gene with the
A slightly different formula, [BCR-abl/(abl – first three (type A or S translocation) or six (type B or L
BCR-abl)] × 100, for the transcript ratio is used translocation) exons of the PML gene joining to exons 2
if the abl primers also amplify the BCR-abl trans- to 6 of the RARA gene (Fig. 13.39).
location.122 In the calculation shown in the text, Test methods similar to those described previously
BCR-abl/abl × 100 yields 0.6%; [BCR-abl/(abl – for BCR-abl translocation are used to detect t(15;17),
BCR-abl)] × 100 yields 0.85%. although there is no current standardization other than
standard curves generated from reference standards.
PML gene RARA gene
1 2 3 4 5 6 2 3 4 5 6
FIGURE 13.39 Location of primers for
PCR analysis of the long (top) and the
short (bottom) translocated genes. The
first three or six exons of the PML gene
1 2 3 2 3 4 5 6 are fused to exon 2 of the RARA gene.
The intron and exon lengths are not
drawn to scale.
Reverse transcriptase PCR and RT-qPCR are most fre- region, and the catalytic domain is interrupted by a
quently used. Primers complementary to sequences in hydrophilic “interkinase” sequence of variable length.
exon 3 or 6 of the PML gene and exon 2 of RARA gen- The fibroblast growth factor receptors (FGFR) repre-
erate products only if the translocation has occurred. The sent the fourth class, which differ from the third class
presence of the translocation product is interpreted as a by having only three immunoglobulin-like domains in
positive result. As with any test of this type, an amplifi- the extracellular region and a short kinase insert in the
cation control is required to avoid false-negative results. intracellular domain. FLT3 is a member of the third class
For quantitative PCR, results normalized to an internal of tyrosine-kinase receptors.
control, using calculations as described previously, yield Particular mutations in the FLT3 gene aberrantly
the most consistent day-to-day results (lowest coefficient activate the FLT3 kinase and predict prognosis in
of variance).127 AML. These mutations include internal tandem dupli-
cations (ITDs) close to the transmembrane domain or
Advanced Concepts point mutations affecting an aspartic acid residue in the
kinase domain (D835 mutations). The ITD can easily be
In the t(15;17) translocation, the reciprocal fusion detected by PCR with primers flanking the potentially
gene RARA/PML is also expressed in 70% to duplicated region. The size of the amplicon observed by
80% of cases of APL. Nonreciprocal events can agarose or capillary gel electrophoresis will increase in
produce either fusion alone as well.128 Although the event of an ITD.130 D835 mutations can be detected
the reciprocal fusion may participate in the tum- by PCR-RFLP, where an EcoRV restriction site is
origenesis process, it does not predict response to destroyed by the presence of the mutation or by RT-PCR
all-trans retinoic acid therapy.129 The presence of with FRET probes.131 In performing these assays, it is
the reverse transcript may be used to confirm the important to have adequate representation of tumor cells
translocation and could be useful in cases where in the specimen to avoid false-negative results from the
the PML/RARA transcript is poorly expressed. presence of an excess of normal cells.
As data from mutation analysis grow, the interpre-
tation of mutations has become more interrelated. For
FMS-Related Tyrosine Kinase 3 (FLT3), 13q12 AML prognosis, it is recommended to interpret FLT3
Four classes of growth factor–receptor tyrosine kinases mutation results with karyotyping and tests for other
have been categorized (see Fig. 13.2). One class, repre- gene mutations, including nucleophosmin (NPM) inser-
sented by the ERBB (EGFR) family, was described in tion mutations and point mutations in the CCAAT/
earlier sections. A second class includes dimeric recep- enhancer-binding protein alpha (CEBPA) and the isoc-
tors such as the insulin growth factor receptor (IGFR) itrate dehydrogenase genes, IDH1 and IDH2. If the
and several proto-oncogenes. Members of the third karyotype is normal with an NPM1 mutation but no
class, including FMS, PDGFR, FLT1, and KIT, display FLT3 ITD, or with a CEBPA mutation, the prognosis is
five immunoglobulin-like domains in the extracellular favorable, similar to that of patients with chromosome
16 inversions or a t(8;21) translocation. With an FLT3

Mutation Spectra
ITD mutation and normal karyotype, the prognosis is
less favorable.132 IDH mutations are potential therapeu- Tumor-suppressor genes and oncogenes are disrupted by
tic targets.133 numerous genetic events. A number of specific abnormal-
ities are associated with particular diseases (Table 13.7).
Janus Kinase 2 (JAK2), 9p24 These chromosome irregularities are targets for diag-
The JAK2 gene codes for a kinase enzyme that phos- nosis of the associated diseases. The growing number
phorylates several Signal Transducer and Activator of of clinically significant variants has driven the use of
Transcription (STAT) gene products, bringing about high-throughput methods such as array analyses and
cellular responses. A high proportion of patients with NGS for more comprehensive information and more
polycythemia vera, essential thrombocythemia, or idio- accurate interpretations.
pathic myelofibrosis carry a dominant mutation causing Many molecular oncology tests have been developed
a valine-to-phenylalanine amino acid substitution at by individual laboratories from published or original
position 617 of the Jak2 protein (V617F). Detection of procedures rather than purchased as a set of reagents or
this mutation aids in the diagnosis of these myeloprolif- in kit form from a commercial source. Often the final
erative disorders. A convenient method of detection is to details of these procedures are determined empirically
use multiplex sequence-specific PCR. In this assay, four so that, in detail, a test procedure can differ from one
primers are used, one forward and one reverse primer laboratory to another. Even after the test procedure is
flanking the region of the mutation, one forward primer established, troubleshooting performed as the procedure
ending at the mutation site complementary to the normal is put into use on a routine basis may further modify
base, and a fourth reverse primer also ending at the site of the procedure. Some reactions that work well for short-
the mutation but complementary to the mutant base. The term research purposes may prove to be less consistent
mutation is revealed as the product of the fourth reverse under the demands of the clinical laboratory setting.
primer and the outer forward primer (see Chapter 8, Even with high-throughput sequencing, the allelic frac-
Fig. 8.18). In some laboratories, white blood cells are tion of mutations/normal sequence and patient charac-
fractionated in order to perform JAK2 testing specifi- teristics (age, gender, type and location of tumor) have
cally on the granulocyte fraction. The mutant/normal to be considered.
ratio is important, based on more recent data suggesting Biotechnology is rapidly developing standard reagent
that the heterozygous/homozygous state of the V617F sets and sequencing panels (primers) for the most
mutation has implications regarding which myelopro- popular tests, but these may also differ from one sup-
liferative disorder is present.134 Furthermore very low plier to another. Furthermore, due to market demands or
levels of V617F may be found in normal individuals. new approaches such as “liquid biopsies,” test reagent
Four mutations affecting exon 12 of JAK2 have kits may be modified or discontinued. If replacement
been identified in some V617F-negative patients: F537- reagents are available, they may not be identical to those
K539delinsL, K539L, H538QK539L, and N542E543del. previously used. Ongoing tests then have to be opti-
These mutations are not located in the kinase domain of mized. This can be a concern where turnaround times
the Jak2 protein, but they do promote higher signaling are critical.
in the cell than does the V617F mutation. Although the It then becomes the responsibility of the technologist
exon 12 mutations are not found in essential thrombo- to perform and monitor tests on a regular basis to main-
cythemia, mutations in the myeloproliferative leukemia tain consistency and accuracy of results. The technol-
(MPL) gene may be present, suggesting a molecular ogist who understands the biochemistry and molecular
differentiation between polycythemia and thrombo- biology of these tests will be better able to respond to
cythemia. These mutations are detected using direct these problems. In addition, with the evolution of the
sequencing, although other methods, such as nonisotopic sciences, a knowledgeable technologist can better rec-
RNase cleavage assay (NIRCA), have been reported ognize significant discoveries that offer the potential for
for JAK2.135 test improvement.
TABLE 13.7 Chromosomal Abnormalities Associated With Leukemias and Lymphomas
Disease Chromosomal Mutation
Pre-B acute lymphoblastic leukemia t(1;19)
Acute lymphocytic leukemia t(4;11), t(11;14), t(9;22), del(12)(p11) or (p11p13) or t(12)(p11), i(17q),
t(9;22), t(12;21), t(8;14), t(2;8), t(8;22), t(11q)
B-cell leukemia t(2;8), t(8;14), t(8;22), t(11;14)
Acute T-lymphocytic leukemia t(11;14), del(9)(p21 or 22)
Acute myeloid leukemia/myelodysplastic syndrome t(11q23)-multiple partners
Acute myeloid leukemia (M2) t(8;21), t(6;9)
Acute promyelocytic leukemia (M3) t(15;17), 14q+
Acute myelomonocytic leukemia (M4) t(11;21), inv(16)(p13q22)
Acute monocytic leukemia (M5) t(9;11), del(11)(q23), t(11q23)-multiple partners
Chronic myelogenous leukemia t(9;22), t(11;22), +8, +12, i(17q)
Acute nonlymphocytic leukemia t(8;21), -Y
Chronic lymphocytic leukemia 14q+, +12, t(14;19), del(11)(q22), del(13q), del(17)(p13)
T-chronic lymphocytic leukemia Inv(14)(q11q32) or t(14;14)(q11;q32)
Burkitt lymphoma t(8;14), t(2;8), t(8;22)
Diffuse large B-cell lymphoma t(3q27), t(14;18), t(8;14)
T-cell lymphoma t(8;14)
Follicular lymphoma t(14;18), t(8;14)
Mantle cell lymphoma t(11;14)
Multiple myeloma t(14q32), 14q+
Myeloproliferative/myelodysplastic disease del(5)(q12q33), -7 or del(7)(q22), +8, +9
Myeloproliferative/myeloblastic disease del(13)(q12 or q14), +21
Hairy cell leukemia 14q+
Waldenström macroglobulinemia 14q+
Mucosa-associated lymphoid tissue lymphoma t(11;18), t(14;18), t(1;14)
Polycythemia vera del(20)(q11.2q13.3)


A 40-year-old woman with a history of non- A 54-year-old woman with thrombosis, a high
Hodgkin’s lymphoma reported to her physician for platelet count (900,000/μL), and a decreased
follow-up testing. Her complete blood count (CBC) erythrocyte sedimentation rate was tested for
was normal, including a white blood cell (WBC) polycythemia vera. Megakaryocyte clusters and
count of 11,000/μL. Morphological studies on a pyknotic nuclear clusters were observed in a bone
bone marrow biopsy and a bone marrow aspirate marrow biopsy. Overall cellularity was decreased.
revealed several small aggregates of mature and In the CBC, white blood cell and neutrophil
immature lymphocytes. Flow-cytometry studies counts were normal. Iron stores were also in the
were difficult to interpret because there were too normal range. A blood sample was submitted to the
few B cells in the bone marrow aspirate specimen. molecular pathology laboratory for JAK2 V617F
No chromosomal abnormalities were detected by mutation analysis. The results were reported as
cytogenetics. A bone marrow aspirate tube was follows:
also sent for molecular analysis—namely, immu-
noglobulin heavy-chain gene rearrangement and
t(14;18) gene translocation analysis by PCR. The
immunoglobulin heavy-chain gene rearrangement
results are shown in lane 4 of the following gel
image:
Agarose gel electrophoresis of SSP-PCR amplicons. Extension

of a JAK2 V617F–specific primer produces a 270-bp band in
addition to the normal 500- and 230-bp bands. Lane 1, molec-
ular-weight markers; lane 2, patient specimen; lane 3, V617F
mutant control; lane 4, normal control; lane 5, reagent blank.
Immunoglobulin heavy-chain gene rearrangement results (left):
lane 1, molecular-weight marker; lanes 2 to 4, patient speci- QUESTIONS:
mens; lane 5, positive control; lane 6, sensitivity; lane 7, nega- 1. What is the source of the two bands in the
tive control; lane 8, reagent blank. t(14;18) gene translocation
normal control in lane 4?
test (right): lane 1, molecular-weight marker; lanes 2 to 6,
patient specimens; lane 7, positive control; lane 8, sensitivity; 2. What is the source of the additional band in the
lane 9, negative control. positive control in lane 3?
QUESTIONS: 3. Does this patient have the JAK2 V617F
mutation?
1. Does this patient have an amplifiable gene
rearrangement?
2. Are the translocation results at right consistent
with those of cytogenetics?
3. What control is missing from the translocation
analysis?
Case Study 13.3

Paraffin-embedded sections were submitted to the for confirmation of the location of tumor cells on the
molecular diagnostics laboratory for p53 mutation sections. These cells were dissected from the slide.
analysis. The specimen was a small tumor (intra- DNA isolated from the microdissected tumor cells
ductal carcinoma in situ) discovered in a 55-year- was screened by SSCP for mutations in exons 4 to 9
old woman. Lymph nodes were negative. Slides of the p53 gene. The results for exon 5 were reported
stained with hematoxylin and eosin were examined as follows:
Polyacrylamide gel electrophoresis with silver-stain detection of p53 exon 5 (left). Lane 1, normal; lane 2, patient; lane 3, normal. Direct
sequencing (right) revealed a C→T (G→A) base change, resulting in an R→H amino acid change at position 175.
QUESTIONS: 3. What further confirmation/information is gained

1. What information is gained from the SSCP results? from direct sequencing?
2. What is the source of the additional band in the
patient SSCP lane?
Case Study 13.4

Colon carcinoma and three polyps were resected in a instability testing. DNA was isolated from tumor
right hemicolectomy of a 33-year-old man. Without cells dissected from four paraffin sections and from
a family history, the man’s age and the location of the patient’s white blood cells. Both were amplified
the tumor warranted testing for HNPCC. Histologi- at the five microsatellite loci recommended by the
cal staining was negative for MSH2 protein. Paraffin National Cancer Institute. The results were reported
sections and a blood sample were submitted to the as follows:
molecular pathology laboratory for microsatellite
Case Study 13.4 (Continued)
MSI analysis of normal cells (N, top row) and tumor cells (T, bottom row) at the five NCI loci.
QUESTIONS: 2. What accounts for the difference in the peak pat-

1. What are the indications for MSI analysis in this terns in the BAT25 and BAT26 loci?
case? 3. How would you interpret the results of this analy-
sis according to NCI recommendations?
Case Study 13.5

A 23-year-old college student was sent to the
student health office with a painful bruise that per- STUDY QUESTIONS
sisted for several weeks. He was referred to the
local hospital, where the physician ordered a bone
1. What are the two important checkpoints in the cell
marrow biopsy and a CBC. His WBC count was
division cycle that are crossed when the regulation
28,000/μL; red blood cells, 2 million/μL; hemo-
of the cell division cycle is affected?
globin 8, hematocrit 20, platelets, 85,000/μL.
Neutrophils were 7%, lymphocytes were 5%,
2. An EWS-FLI-1 mutation was detected in a solid
and blasts were 95%. The pathologist who exam-
tumor by RT-PCR. Which of the following does
ined the bone marrow biopsy requested flow-
this result support?
cytometry analysis of the aspirate. The cells
expressed 84% CD20, 82% CD34, 92% HLA-DR, a. Normal tissue
and 80% CD10/CD19. The results of these tests b. Ewing sarcoma
indicated a diagnosis of acute lymphoblastic leu- c. Inherited breast cancer
kemia. A blood specimen was sent to the cyto- d. Microsatellite instability
genetics laboratory for chromosomal analysis.
Twenty metaphases examined had a normal 46,XY 3. Mutation detection, even by sequencing, is not
chromosomal complement. Interphase FISH was definitive with a negative result. Why?
negative for t(9;22) in 500 nuclei.
4. A PCR test for the BCL-2 translocation is
QUESTIONS:
performed on a patient with suspected follicular
1. What is the significance of the cytogenetic test lymphoma. The results show a bright band at about
results? 300 bp for this patient. How would you interpret
2. What other molecular abnormalities might be these results?
present in this type of tumor?
5. Which of the following misinterpretations would d. PCR results are not accepted by the College of
result from PCR contamination? American Pathologists.
a. False positive for the t(15;17) translocation
10. Interpret the following results from a translocation
b. False negative for the t(15;17) translocation
assay.
c. False negative for a gene rearrangement
M 1 2 3 Pos Sens Neg Blank
6. After amplification of the t(12;21) breakpoint

by qRT-PCR, what might be the explanation for
each of the following observations? (Assume that
positive and amplification controls and a reagent
blank control are included in the run.)
a. The gel is blank (no bands, no molecular-weight Target
standard).
b. Only the molecular-weight standard is Amp control
visible.
c. The molecular-weight standard is visible; there Are the samples positive, negative, or
are bands in every lane at 200 bp, even in the indeterminate?
reagent blank lane. Sample 1:
Sample 2:
7. What is observed on a Southern blot for gene Sample 3:
rearrangement in the case of a positive result?
a. No bands 11. Which of the following predicts the efficacy of
b. Germline bands plus rearranged bands EGFR tyrosine-kinase inhibitors?
c. Smears a. Overexpression of EGFR protein
d. Germline bands only b. EGFR-activating mutations
c. Patient gender
8. Cyclin D1 promotes passage of cells through the d. Stage of disease
G1-to-S checkpoint. What test detects translocation
of this gene to chromosome 14? 12. What is the advantage of macrodissection in
a. t(14;18) translocation analysis (BCL2, IGH) testing for tumor-specific molecular markers
b. t(15;17) translocation analysis (PML/RARA) from paraffin-embedded formalin-fixed tissue
c. t(11;14) translocation analysis (BCL1/IGH) sections?
d. t(8;14) translocation analysis (MYC/IGH)
13. Why are KRAS- and BRAF-activating
mutations almost always exclusive of one
9. Why is the Southern blot procedure superior to
another?
the PCR procedure for detecting clonality in some
cases?
14. What enzyme is responsible for continued
a. Southern blot requires less sample DNA than sequence changes in the immunoglobulin
does PCR. heavy-chain gene variable region after gene
b. The PCR procedure cannot detect certain gene rearrangement has occurred?
rearrangements that are detectable by Southern
blot. 15. Why are translocation-based PCR tests more
c. Southern blot results are easier to interpret than sensitive than IgH, IgL, or T-cell receptor gene-
PCR results. rearrangement tests?
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Chapter 14
DNA-Based Tissue Typing
Outline Objectives
THE MHC LOCUS 14.1 Describe the structure and function of the major
HLA POLYMORPHISMS histocompatibility (MHC) locus.
HLA Nomenclature 14.2 Use HLA nomenclature to identify alleles.
MOLECULAR ANALYSIS OF THE MHC 14.3 List the human leukocyte antigens (HLAs) that are
Serological Analysis encoded by the MHC locus, and explain their role
HLA Typing in tissue engraftment and rejection.
Screening 14.4 Compare and contrast the levels of typing
Crossmatching resolution that are achieved by different laboratory
Mixed-Leukocyte Reaction methods.
Protein Gel Electrophoresis 14.5 Describe the laboratory methods used to identify
DNA-Based Typing HLAs by serology testing.
Sequence-Specific Oligonucleotide Probe Hybridization 14.6 Describe the DNA-based testing methods used for
Sequence-Specific PCR the identification of HLAs.
Sequence-Based Typing 14.7 Explain how combining different test methods
Other DNA-Based Methods to identify HLAs increases resolution and resolves
Combining Typing Results ambiguities.
HLA Test Discrepancies 14.8 Relate the use of HLA typing for confirming
Coordination of HLA Test Methods disease diagnosis and predisposition.
ADDITIONAL RECOGNITION FACTORS
Minor Histocompatibility Antigens
Nonconventional MHC Antigens
Killer Cell Immunoglobulin-Like Receptors
MHC DISEASE ASSOCIATION
SUMMARY OF LABORATORY TESTING
417
The major histocompatibility complex (MHC) is a transfused persons contained antibodies that agglu-
group of genes located on the short arm of chromosome tinated leukocytes. This discovery led to serological
6. In humans, the MHC gene products are called human typing methods that originally identified two polymor-
leukocyte antigens (HLAs). The HLAs were named for phic gene loci, HLA-A and HLA-B, followed soon after
their role in the rejection of transplanted organs. Kidney, by the identification of HLA-C and other genes. A test
heart, liver, lungs, skin, pancreas, corneas, blood, bone for typing these loci was designed from the observation
marrow, and hematopoietic stem cells can be transplanted that large immature mononuclear cells proliferated if
from one human to another. Transplanted organs, except lymphocytes from unrelated individuals were mixed and
in the case of identical twins, are allografts, indicating cultured together.1,2 The results from this mixed lym-
genetic differences between the donor of the organ and phocyte culture (MLC) reaction did not always agree
the recipient. When a transplant is performed, compati- with the results of serological typing, however. The dis-
bility (matching) of the HLA of the organ donor and the crepancy was partly resolved by the discovery of addi-
recipient increases the chance for a successful engraft- tional genes comprising the HLA-D locus.3,4
ment that will function for several years. If the donor
and recipient are not HLA-matched, then the recipient’s Histooricaal Higghlligghtts
immune system (primarily mediated by T lymphocytes
as well as B lymphocytes) will recognize the donor The genetic contribution to transplant rejection
organ as nonself (foreign) and will mount an immune was first proposed in 1927 when Bover observed
response against the organ, resulting in its destruction, that skin transplants between identical twins
loss of function, and rejection by the recipient. were not rejected like those from genetically dis-
The role of the clinical laboratory is to evaluate the tinct individuals.5 The genes involved were first
HLAs of potential donors and recipients and to aid in described in mice by Gorer.6 Snell7 used mouse
the prediction of successful engraftment and the avoid- cell lines to further define a genetic locus, which
ance of graft rejection and graft-versus-host disease he called H for histocompatibility. Gorer referred
(GVHD). Graft rejection results in the failure of the to the gene products of this locus as antigen II,
donor organ due to an immune reaction of the recipi- and the combined term H-2 was subsequently
ent against it. GVHD is the reciprocal of graft rejection used for the MHC locus in mice.
whereby immunocompetent cells in the donor organ rec-
ognize recipient cells as foreign and attack and destroy
the recipient cells, resulting in significant morbidity and HLAs are divided into three classes (I, II, and III), all
potential mortality to the recipient. The process of HLA encoded by a gene complex located on chromosome 6p
identification utilizes methods targeting cell-associated (Fig. 14.1). The MHC locus includes genes other than
antigens as well as serum antibodies to “nonself” cells those that code for the HLA. Cytokine genes and genes
and tissues. Most tissue typing, as HLA identification is encoding tumor necrosis factor α (TNF-α) and tumor
commonly called, was performed initially by serological necrosis factor β (TNF-β) are located inside of the main
methods using antibodies to the different HLAs found HLA complex.8
in the human population. Increasingly, however, DNA In addition to the main MHC locus, gene regions
typing methods are being implemented for this purpose, extending beyond the HLA-DP genes toward the cen-
increasing the sensitivity and specificity of the typing tromere and HLA-F toward the telomere comprise the
procedure. extended MHC locus (xMHC). The xMHC locus
covers 8 Mb and includes the hemochromatosis gene
HLA-F (also called HFE), the farthest telomeric gene
THE MHC LOCUS in the complex.9,10 The most centromeric locus of the
extended MHC is the tapasin region. Tapasin is required
The human MHC locus was discovered in the early for antigenic peptide processing.11 Some genes that are
1950s. Investigators independently noted that blood associated with disease conditions, such as HFE linked
from women who had borne children or from previously to the MHC locus, are the basis for the association of
Chapter 14 • DNA-Based Tissue Typing 419
0 1 2 3 4 Mb
DP DQ DR
TNF
Chromosome 6 ␤␣␤␣ ␤␣ ␤␤ ␤ ␤ ␣ ␣ ␤ B C A
HLA: Class II Class III Class I
FIGURE 14.1 The MHC locus on chromosome 6 covers about 4 Mb of DNA, depending on the individual. Class I genes are 3 to
6 kb long, and class II genes are 4 to 11 kb in length. TNF-α and TNF-β are not part of the polymorphic HLA system.
TABLE 14.1 Genes of the Major Histocompatibility Locus
MHC Region Gene Products Tissue Location Function
Class I HLA-A, HLA-B, HLA-C All nucleated cells Identification and destruction of abnormal
or infected cells by cytotoxic T cells
Class II HLA-D B lymphocytes, monocytes, Identification of foreign antigen by helper

macrophages, dendritic cells, activated T cells
T cells, activated endothelial cells, skin
(Langerhans’ cells)
Class III Complement C2, C4, B Plasma proteins Defense against extracellular pathogens
Cytokine genes TNF-α, TNF-β Plasma proteins Cell growth and differentiation
particular disease states with HLA type.12 The role of the cells, and macrophages. As illustrated in Figure 14.2,
immune system in autoimmune diseases and susceptibil- class I molecules consist of a long (heavy) chain of 346
ity to infections also links HLA type to disease. amino acids (44 kD) associated with a 12-kD peptide,
Despite the discovery of the MHC gene products β-2 microglobulin, containing 99 amino acids that are
as mediators of transplant rejection, recognition of not encoded in the MHC. These two chains are associ-
genetically different (“nonself”) organs and tissues is ated with one another on the cell surface by noncovalent
not the main function of these glycoproteins. HLAs bonds. The class I heavy chain displays short branched-
appear on the surface of cells of the immune system, chained sugars, making this molecule a glycoprotein.
allowing cell–cell communication during immune func- The heavy chain is also a transmembrane polypeptide,
tions. Immune reactions involve and are restricted by anchoring the complex at the surface of the cell. Class II
interactions between T lymphocytes (cells involved in molecules consist of two transmembrane polypeptides,
cell-mediated immunity), B lymphocytes (antibody- an α chain with three domains, α1, α2, and α3, and a β
producing cells), and the MHC molecules. chain with two domains, β1 and β2. The two polypep-
The gene products of the MHC, class I, II, and III tides associate, forming a groove between the α1 and β1
proteins, are present in different amounts on different domains that will hold fragments of antigen that have
tissues (Table 14.1). Class I and II are the strongest been engulfed and processed by the cell (extracellular
antigens expressed on cells. Class I molecules (des- antigens). In contrast, antigens bound to class I mol-
ignated as A, B, or C) are expressed on all nucleated ecules (where the peptide-binding domain is formed
cells, whereas class II molecules (designated as D) are between the α1 and α2 domains) are generated from the
only expressed constitutively on “professional antigen- processing of macromolecules synthesized within the
presenting cells,” such as B lymphocytes, dendritic cell (intracellular antigens).
HLA POLYMORPHISMS
Outside of cell
Class II Class I Genes of the MHC are the most polymorphic genes of
␣1 ␤1 ␣2 ␣1
the human genome. Polymorphisms in this locus were
first defined phenotypically by acceptance or rejection
S S
S S of tissue or by reaction with defined antibodies (sero-
logical typing). Molecular typing methods reveal HLA
␣2 ␤2 ␣3
polymorphisms as base changes in the DNA sequence
(Fig. 14.3). The changes range from a single base pair
S
S
S S
S
S
S
S (single nucleotide polymorphisms) to loss or gain of
entire genes. A particular sequence, or version, of an
␤2-
HLA gene is an allele of that gene. The HLA type is the
microglobulin
collection of alleles detected by phenotypic or genotypic
typing methods.
Cell Thus, each HLA gene can differ in sequence from any
membrane individual to another, except for identical twins. A set of
particular alleles on the same chromosome is a haplo-
Cytosol type (Fig. 14.4). These alleles are inherited together as
a block of chromosomal sequence, unless a rare recom-
␣ chain ␤ chain ␣ chain
bination event within the region separates the alleles. An
HLA haplotype is, therefore, the combination of poly-
morphic sequences or alleles in the HLA gene regions.
FIGURE 14.2 Class II (left) and class I (right) polypeptides. Polymorphisms are concentrated in exons 2 and 3 of
Class II antigens consist of two chains, α and β. Class I anti- the class I genes and in exon 2 of the class II genes.
gens consist of a heavy chain and a light chain associated These exons code for the amino acids that interact with
together with a molecule of β-2 microglobulin.
antigenic peptides, affecting the recognition of nonself
peptides. Molecular methods target these exons in
HLA-A, HLA-B, and HLA-C class I genes and mostly
HLA-DRB class II genes. Other areas including introns
have been investigated for potentially useful alleles.15
HLA Nomenclature
Advanced Concepts
Polymorphisms are alterations in DNA and/or protein
Class I and II molecules present fragments of sequences shared by 1–2% of a defined population. For-
antigens, usually about nine amino acids long, to mally, alterations present at lower frequencies are called
T lymphocytes. Class I and II molecules vary from mutations or variants. Structurally, mutations and poly-
one another (are polymorphic), sometimes by a morphisms are the same thing, changes from a consensus
single amino acid. Due to these polymorphisms, amino acid or nucleotide sequence. Alleles are the dif-
different HLA molecules (HLA types) vary in their ferent versions of the gene. The polymorphic nature of
efficiency of binding antigen fragments, result- the MHC, therefore, means that there are multiple alleles
ing in a range of immune responses to a given of each HLA gene present in the human population.
antigen. This distinction can affect the symptoms These alleles differ by nucleotide sequence at the DNA
of disease, for example, the likelihood of persons level (polymorphisms) and by amino acid sequence and
of a particular HLA type infected with HIV to antigenicity at the protein level. Polymorphisms arise
develop full immunodeficiency.13,14 mostly as a result of gene-conversion events and rare
chromosomal recombinations. Each person will have a
CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA
CGG GCC GCG GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GAG AGC TTC ACA
A CGG GCC GCC GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GNN NNN NNN NNN
CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA
– – – – – – – – – – – – – – – – – – – –T – – – – – – – – – – – – – – – – – – – C– –TG – – – – – – – – – – – –
B – – – – – – – – C – – – – – – – – – – –T – – – – – – – – – – – – – – – – – – –C– –TG – * * *** *** ***
FIGURE 14.3 (A) DNA polymorphisms in the HLA-DRB1 gene. The initial sequence (DRB1*01:01) is written at the top of the
panel. Aligned underneath are two alleles of this region, DRB1*01:02 and DRB1*01:03. The bases in color are those that differ
from DRB1*01:01. N indicates unknown or unsequenced bases. (B) A different way of presenting the alleles. A dash indicates
identity to the consensus sequence. Only the polymorphic bases are written. The asterisk indicates unknown or unsequenced bases.
Parental genotypes
A24 A30 A1 A6
Cw1 Cw3 Cw1 Cw7 Alleles

Haplotype
B14 B7 B12 B44
X
DR14 DR15 DR5 DR14
Offspring
A24 A6 A1 A30 A6 A30 A24 A1
Cw1 Cw7 Cw1 Cw3 Cw7 Cw3 Cw1 Cw1

B14 B44 B12 B7 B44 B7 B14 B12
FIGURE 14.4 A haplotype is the combina-

tion of alleles that are inherited together. In
this example, parental genotypes (top) can DR14 DR14 DR5 DR15 DR14 DR15 DR14 DR5
produce four possible genotypes in the off-
spring (bottom).
particular group of HLA alleles inherited from his or her defined alleles: a number follows the gene region name;
parents. The maternal and paternal HLA antigens are for example, HLA-B51 denotes HLA-B antigen 51
expressed codominantly on cells. (defined by reaction to a known antibody). Number des-
HLA alleles were first defined at the protein level ignations of new alleles of a previously defined allele
by antibody recognition (serologically). A standard with broad specificity (parent allele) are followed by the
nomenclature for expressing serologically defined anti- number of the parent allele in parentheses. For example,
gens was established by the World Health Organization HLA-A24(9) denotes the HLA-A antigen 24 from parent
(WHO) Nomenclature Committee for Factors of the antigen 9. The derived antigens are called split specific-
HLA System. In this system, HLA refers to the entire ities. Additional antigens have been defined by reactions
gene region, and A, B, and D refer to the particular loci; between known antigens and serum antibodies (antise-
for example, HLA-A, HLA-B, or HLA-D. The HLA-D rum reactivity). The number of HLA alleles increase as
locus consists of subregions P, Q, M, O, and R, termed new specificities are defined.
HLA-DP, HLA-DQ, HLA-DM, and so forth. Each of With the introduction of molecular biology techniques
these subregions consists of genes that code for either in the 1980s, HLA typing at the DNA level required
an α- or β-chain polypeptide; for example, the first nomenclature for specific DNA sequences.16,17 Many
β polypeptide is encoded in the HLA-DRB1 gene new alleles have been and are currently being defined
(see Fig. 14.1). A small w is included in HLA-Cw, at the DNA level. A revised nomenclature is used for
HLA-Bw4, and HLA-Bw6 allele nomenclature. The denoting alleles defined by DNA sequence. The alleles
w denotation was originally a designation of alleles in are named in sequential order as they are discovered.
“workshop” status or found in high prevalence in the The gene name, such as HLA-DRB1, is followed by
population. Workshop designation is no longer required an asterisk or separator (*), the allele sequence family
for the HLA-C locus; however, the w was retained for number (allele group or type), and then a number for the
HLA-C alleles to distinguish them from the C desig- specific allele (DNA sequence) separated by a colon or
nation used for complement genes. The w is retained field separator. For example, A*25:03:07 is the seventh
with HLA-Bw4 and HLA-Bw6, which are considered polymorphic HLA protein of the third specific allele, 03,
“public” (high-prevalence) antigens. of the HLA-A*25 family of alleles. Additional infor-
A list of the serologically defined alleles of the HLA mation not routinely used in identifying alleles is also
genes accepted by the WHO is shown in Table 14.2. part of their nomenclature. Silent mutations (changes
The WHO official nomenclature refers to serologically in the DNA sequence that do not change the amino
TABLE 14.2 Serologically Defined HLA Specificities*
HLA-A HLA-B HLA-C HLA-DR HLA-DQ HLA-DP
A1 B5 Cw1 DR1, DR103 DQ1 DPw1
A2, A203, A210 B51(5), B5102, B5103 Cw2 DR2 DQ5(1) DPw2
A3 B52(5) Cw3 DR15(2) DQ6(1) DPw3
A9 B7, B703 Cw9(w3) DR15(2) DQ2 DPw4
A23(9) B8 Cw10(w3) DR3 DQ3 DPw5
A24 (9), A2403 B12 Cw4 DR17(3) DQ7(3) DPw6
A10 B44(12) Cw5 DR18(3) DQ8(3)
A25(10) B45(12) Cw6 DR4 DQ9(3)

TABLE 14.2 Serologically Defined HLA Specificities (Continued)
A26(10) B13 Cw7 DR5 DQ4
A34(10) B14 Cw8 DR11(5) DQ3
A66(10) B64(14) Cw10(w3) DR12(5)
A11 B65(14) DR6
A19 B15 DR13(6)
A74(19) B62(15) DR14(6), DR1403, DR1404
A68(28) B63(15) DR7
A69(28) B75(15) DR8
A29(19) B76(15) DR9
A30(19) B77(15) DR10
A31(19) B16 DR51
A32(19) B38(16) DR52
A33(19) B39(16), B3901, B3902 DR53
A36 B17 DR16(2)
A43 B57(17)
A80 B58(17)
A28 B18
A74 (19) B21
B49(21)
B50(21)
B22
B54(22)
B55(22)
B56(22)
B27, B2708
B35
B37

TABLE 14.2 Serologically Defined HLA Specificities (Continued)
B40, B4005
B60(40)
B61(40)
B41
B42
B46
B47
B48
B53
B59
B67
B70
B71(70)
B72(70)
B73
B7801
B81
Bw4
Bw6
B51(5), B51:02, B51:03
B52(5)
B57(17)
B58(17)
B62(15)
B63(15)
B64(15)
B65(15)
*Alleles are listed as broad antigen groups, followed by split antigens for that group, with the broad antigen number in parentheses. Associated antigens, such as
B40 and B4005, are listed together.
acid sequence), also called synonymous changes, are

designated by a number following the specific allele 15:57/15:60, in any HLA gene is designated RDX
number. For example, A*03:01:02 indicates a synony- so that B*15:01/15:01N, 15:11/15:15, 15:33/15:34,
mous allele, 02, of the first specific allele, 01, from the 15:57/15:60 = B*15RDX. Allele-specific codes
HLA-A*03 family of alleles. A fourth number designa- are used for allele combinations that include more
tion indicates that the synonymous change results from a than one serological family or that contain an N,
polymorphism in intronic sequences beyond the coding L, or S expression.
regions (exons) of the genes. Thus, A*26:07:01:01 is the
01 subtype allele of the HLA-A*26:07 allele, the second
01 indicating that the polymorphism is in an adjacent
intron. Ambiguity is the recognition of two or more antigens
Optional suffixes can also be added to the allele name. by the same antibody, or cross-reaction, so that the
The letter N following the specific allele number indi- exact allele cannot be called. Ambiguity is designated
cates a null allele, or phenotypic absence of that antigen. by a slash (/) between the possible allele numbers or
For example, B*13:07N denotes the 07 allele of the an en dash (–) for a series of alleles in which the first
13-allele family in the HLA-B locus at the DNA level. and last allele are named. For example, if a typing test
At the protein level, however, the encoded protein does results in either B*07:33 or B*07:35, the notation is
not react with any antibody. The B*13:07N null allele is B*07:33/B*07:35. If a typing test indicates that the allele
due to a 15-bp deletion in the HLA-B gene. Null alleles is either B*07:33, B*07:34, B*07:35, or B*07:36, the
can also result from nonsense, frameshift, splice site, or designation is B*07:33–B*07:36. Ambiguity also arises
other premature stop mutations that prevent translation from the inability of some typing methods to assign het-
of the amino acids destined to bind to antibody. erozygous alleles to one or the other chromosome. A
Other descriptive designations are less frequently combination of methods may be used to resolve ambi-
used, including L, S, Q, A, C, X, and ?. The letters L and guities. Family studies are also helpful in this regard.
S indicate poor expression of the allele at the cell surface Resolution is the level of detail to which the allele
or a soluble allele, respectively. Q is used to indicate is determined. Low resolution identifies broad allele
questionable allele expression based on the effect of types or groups of alleles. A typing of A*26 is low
the DNA mutation on expression of other alleles. If no resolution, which can be determined at the serological
other information is available, an A indicates aberrant level. Typing methods that detect specific alleles in addi-
expression without confidence that the allele is actually tion to the identification of all serological types are at
expressed. C indicates allele expression in the cyto- medium resolution. The typing result A*26:01/A*26:05/
plasm, rather than on the cell surface. (C and A have A*26:10/A*26:15 is medium resolution. High-resolution
yet to be used in allele names.) An X notation indicates typing procedures can discriminate between almost all
that resolution of the allele was not done, for example, specific alleles. A*26:01 is high resolution determined
B*08X. A question mark (?) indicates that the resolution by DNA analysis. A range of methods, from serologi-
was not clear: B*08?. cal typing to direct DNA sequence analysis, affords the
laboratory a choice of low-, medium-, or high-resolution
typing. Whereas low resolution is adequate for solid
Advanced Concepts organ transplantation typing, bone marrow or stem cell
transplants require high-resolution methods.
The National Marrow Donor Program assigns
alphabetical allele codes, such as AD or RJH,
to allele combinations from submitted requests. MOLECULAR ANALYSIS OF THE MHC
Generic codes can be used with several loci and
allele families. For example, the combination of There are over 17,000 HLA and related alleles described
alleles, 15:01/15:01N, 15:11/15:15, 15:33/15:34, in the International ImMunoGeneTics Project (IMGT)
IPD-IMGT/HLA Database (Table 14.3). Genetic
TABLE 14.3 Number of HLA Alleles Identified Serologically and by DNA Sequence*
Gene Serology Genetic Gene Serology Genetic
Class I HLA-DQA1 94
HLA-A 28 4,081 HLA-DQB1 9 1,178
HLA-B 29 4,950 HLA-DMA 7
HLA-C 10 3,685 HLA-DMB 13
Class II HLA-DPA1 64
HLA-DRA 7 HLA-DPB1 963
HLA-DRB1 18 2,146 HLA-DOA 12
HLA-DRB2 1 HLA-DOB 13
HLA-DRB3 1 152 Extended MHC
HLA-DRB4 1 74 HLA-E 6
HLA-DRB5 1 55 MICA 107
HLA-DRB6 3 MICB 42
HLA-DRB7 2 TAP1 6 12
HLA-DRB8 1 TAP2 4 12
HLA-DRB9 6
*The numbers represent the number of named alleles for each gene as listed by IMGT (http://www.ebi.ac.uk/ipd/imgt/hla). The World Marrow Donor Association
Quality Assurance and Working Group on HLA Serology to DNA Equivalents publishes a comprehensive dictionary of antigen and allele equivalents.
(DNA-based) typing concentrated in the HLA-A, donors, the extent of HLA-type matching between donor
HLA-B, HLA-C, and HLA-DRB1 genes has identified and recipient predicts the long-term survival of the donor
alleles that far outnumber the serological alleles defined organ in the recipient. Furthermore, because disease
for these genes. Over 400 new nucleotide sequences genes are located in and around the MHC locus, certain
were defined in just 2 years, from 2002 to 2004. The HLA gene alleles are linked to disease, affording another
WHO Nomenclature Committee devised rules for the aid in diagnosis or prediction of disease phenotypes.
submission of new alleles for official numerical designa- There are three approaches to the analysis of HLA
tion.18 Allele sequences are stored in the GenBank, the alleles in the HLA laboratory: typing, screening, and
European Molecular Biology Laboratory, and the DNA crossmatching. Typing is the initial identification of the
Data Bank of Japan databases. A list of newly reported HLA alleles of a specimen through protein or DNA-
alleles is published monthly in the journals Tissue Anti- based methods. Typing may be used both to define HLA
gens, Human Immunology, and the International Journal haplotypes and to look for specific HLA types that are
of Immunogenetics. A comprehensive dictionary of linked to disease states. Screening is the detection of
antigen-DNA sequence allele equivalents is published anti-human antibodies in serum that match known HLA
periodically.19 alleles. Crossmatching is the more specific screening of
Identification of alleles in the laboratory serves recipient sera for antibodies against antigens displayed
several purposes. In addition to the selection of organ by potential organ donors.
Serological Analysis ethnic populations or antigens of high prevalence in par-

ticular geographical areas. As the antibody preparations
Traditionally, HLA typing for organ transplantation was are used repeatedly, the antigen binding characteristics
performed serologically; that is, by antigen–antibody of the various antibodies are recognized and recorded.
recognition. Although serological testing yields only Experienced technologists have detailed documentation
low-resolution typing results, there are some advantages of antibody panels, including which antibodies bind
to these methods. Serological typing is a relatively rapid antigen well and which antibodies bind less strongly.
method that reveals immunologically relevant epitopes. To begin the typing procedure, different antibod-
Also, these studies can be used to resolve ambiguities ies are placed in each well of the typing tray. Donor
or confirm null alleles detected by other methods. Sero- or recipient lymphocytes to be typed are distributed to
logical tests include HLA phenotype determination, in the wells. Cross reactivity is assessed by the uptake of
which patient cells are tested with known antisera (HLA trypan blue or eosin red dye in cells that have been per-
typing), and screening of patient sera for anti-HLA meabilized due to reaction with the antibody and with
antibodies. complement that is activated by the antigen–antibody
complexes (Fig. 14.6). Cytotoxicity is scored by the
HLA Typing
estimated percentage of cells in a well that have taken
Lymphocytes are HLA-typed using the complement- up the dye. The American Society for Histocompatibility
dependent cytotoxicity (CDC) test (Fig. 14.5).20 In this and Immunogenetics (ASHI) developed guidelines for
procedure, multiple alleles are determined using a panel the numerical description of the observed cytotoxicity
of antibodies against known HLA types. These anti- (Table 14.4). High cytotoxicity (reading >6) in a well of
bodies are prepared from cell lines or from donors with the plate indicates that the cells being tested have cell
known HLA types. Plates preloaded with antibodies surface antigens matching the known antibody in that
(typing trays) are commercially available. Alternatively, well. Because reading is somewhat subjective, it is rec-
some laboratories construct their own antibody panels. ommended that trays be read by at least two technolo-
The collection of antibodies can be modified to represent gists independently. An example of partial results from
Antigen Complement Dead cell

Lymphocyte
Antibodies
+
Plasma Positive reaction

to antibody
Leukocytes
and platelets
Erythrocytes
+
Blood Negative reaction

to antibody
FIGURE 14.5 Crossmatching to known antibodies is performed on lymphocytes (buffy coat, left) in a 96-well plate format where
each well contains different known antibodies. If the antibody matches the cellular antigen (positive reaction, top), complement-
dependent cytotoxicity will occur, and the dead cell will take up stain (green). If the antibody does not match the cellular antigen,
there is no cytotoxicity.
TABLE 14.4 Expression of CDC TABLE 14.5 Example of Results From a CDC Assay
% Dead or Lysed (dyed) Interpretation Score Antibody Score*
0–10 Negative 1 A2, A28, B7 8
11–20 Doubtful negative 2 A2, A28 6
21–50 Weak positive 4 A10 1
51–80 Positive 6 A10, A11 1
81–100 Strong positive 8 B7, B42 8
Unreadable 0 B7, B27 8
B7, B55 8
B44, B45, B21 6
B44, B45 8
B44 8
B45 1
*Scores are 1–8, depending on the percentage of dyed cells observed.
generated cytotoxicity, supporting the determination of

an A28, B44 haplotype.
CREG matching or residue matching (determined
from the amino acid sequences of the antigens) is con-
sidered for kidney transplant screening in order to define
the spectrum of HLA-specific antibodies more precisely.
Some epitopes are more important than others with
respect to organ rejection. Therefore, certain mismatches
FIGURE 14.6 Cells stained for cytotoxicity. Dead cells take are allowable if the critical epitopes match.
up dye, and live cells remain transparent. (Photo courtesy of Dr.
Andres Jaramillo, Rush University Medical Center.) Screening
Successful organ transplant depends on the minimal
a CDC test is shown in Table 14.5. The results indicate reaction of the recipient immune system to the antigens
that the tested cells are HLA-type A28, B44. of the donor organ. Normal sera do not have antibodies
Each HLA antigen has multiple epitopes, some against human antigens, termed anti-human antibodies
unique to that HLA antigen and some cross-reactive or alloantibodies. Persons who have had a previous organ
epitope groups (CREGs) shared by other HLA anti- transplant, blood transfusions, or pregnancies, however,
gens. The CDC test can be performed with private will have anti-human antibodies (termed humoral sen-
antibodies, those that bind to one specific HLA type, sitization) that may react against a new donor organ.
or antibodies that bind to CREG (or “public” antigens). The chance of a successful transplant is improved by
Analysis of antibodies with shared specificities aids in defining the specificity of the alloantibodies and select-
narrowing the specificity, as illustrated in Table 14.5. ing a suitable organ that does not have HLA antigens
As shown, all antibodies with A28- or B44-specificity corresponding to the antibodies in the patient’s sera.21
Humoral sensitization and the identity of alloantibodies applied, and the antibody-bound beads are detected by
present in the recipient serum are determined in a flow cytometry. The advantages of this method over the
modified version of the CDC assay using the patient’s CDC test are that the reaction is performed in a single
serum as the source of antibodies and reference lympho- tube and there is less subjectivity in the interpretation of
cytes of known HLA types prevalent in the general pop- results. Because this test uses pooled antigens, however,
ulation. The reference lymphocytes are defined by their it can detect the prevalence of anti-human antibodies
recognition of panel reactive antibodies (PRAs). This in the test serum, but it cannot identify which specific
test against PRA estimates the percentage of the general antibodies are present. A negative result does preclude
population with whom the patient will cross-react. further alloantibody assessment. A variation of this
The percentage of the panel of lymphocytes killed by method developed commercially utilizes beads with
the sera is referred to as %PRA. Patients with %PRA their own internal fluorescence. By conjugating known
activity of more than 50% are considered to be highly antigens to beads of different internal fluorescence, the
sensitized; finding crossmatch-negative donors is more positively reacting antibodies can be specifically identi-
difficult in these cases.22 fied while still performing the test in the same tube.
Screening of sera with microparticles (beads) is per-
formed in laboratories with flow-cytometry capabil- Crossmatching
ity (Fig. 14.7). For this method, the beads are coupled The CDC test is also used for crossmatching potential
to pools of antigens derived from cell lines of defined organ donors and recipients. For crossmatching, recip-
HLA types. The beads are then exposed to test serum, ient serum is the source of antibodies tested against
and those beads carrying antigens that are recognized donor lymphocytes (Fig. 14.8). If the recipient serum
by antibodies present in the test serum will bind to kills the donor lymphocytes, it is a positive crossmatch
those antibodies. After removal of unbound antibodies, and contraindication for using the crossmatched donor.
a fluorescently labeled secondary reporter antibody is
Serum antibody Advanced Concepts
Fluorescent
reporter
More detailed crossmatch information is achieved
Antigen
antibody by separate analysis of donor B and T lympho-
Bead
cytes. Unactivated T cells display class I anti-
gens, and B cells display both class I and class
II antigens. Therefore, if B cells cross-react with
the serum antibodies and T cells do not, the serum
A Wash antibodies are likely against class II antigens.
Other methods used for crossmatching include

variations on the lymphocytotoxicity assay and non-
lymphocytotoxic methods that utilize flow cytome-
try, such as the bead arrays just described. Alternative
B
methods also include enzyme-linked immunosorbent
FIGURE 14.7 Detection of serum antibodies using bead assay (ELISA) using solubilized HLA antigens. ELISA
arrays. In this illustration, separate preparations of beads are can be used to monitor the change in antibody produc-
conjugated to two different known antigens. The patient serum tion over time or humoral sensitization developing after
tested contains an antibody to the antigen on the beads in (A)
the transplant.
but not the antigen on the beads in (B). A secondary antibody
targeting the bound serum antibody generates a fluorescent Mixed-Leukocyte Reaction
signal detected by flow cytometry. If a matching antibody is
not present in the test serum as in (B), no antibody will be T lymphocytes are primarily responsible for cell-
bound. mediated organ rejection. The mixed leukocyte culture or
Recipient serum
Antigen Complement Dead cell
Positive reaction
Lymphocytes to antibody
from organ donor
of known HLA type
FIGURE 14.8 In the crossmatch by

CDC assay, the recipient serum is the
source of antibodies to type lympho-
+
cytes from potential organ donors. The
antibodies in the recipient serum can be
Negative, no reaction identified if the HLA type of the lympho-
to antibody cytes is known.
mixed lymphocyte reaction (MLR) is an in vitro method DNA-Based Typing

used to determine T-cell cross reactivity between donor
Typing, screening, and crossmatch analysis are criti-
and recipient. The MLR assay measures the growth of
cal for the selection of potential donors and successful
lymphocytes activated by cross reactivity as an indica-
engraftment. Methods differ in sensitivity. The choice
tion of donor–recipient incompatibility. MLR can also
and design of the method used, therefore, will affect the
test for cell-mediated cytotoxicity and cytokine produc-
ability to predict rejection risk.
tion, by either the donor or recipient lymphocytes, so
Limitations of serological and protein-based methods
it can be used to predict GVHD as well as recipient-
have led to the development of more refined DNA
mediated transplant rejection. For this test, cells must
typing with higher powers of resolution, especially for
be incubated together for several days. Cell activation
bone marrow transplantation. One of the first DNA
and growth are assessed by uptake of 3H-thymine. Even
methods for molecular typing was restriction fragment
though the MLR is more likely to detect HLA mis-
length polymorphism (RFLP) analysis by Southern
matches than serology techniques, the time and techni-
blot to identify HLA class II alleles. Studies showed
cal demands of the MLR precluded its routine use for
that kidneys matched by RFLP typing survived longer
pretransplant histocompatibility testing in the clinical
than those matched by serological typing. Just as with
laboratory.23
other applications of molecular testing, the development
of amplification and direct sequencing methods greatly
Protein Gel Electrophoresis
advanced the analysis of HLA polymorphisms at the
The protein products of the HLA genes can be distin- DNA sequence level.
guished by mobility differences in one-dimensional gel DNA typing focuses on the most polymorphic loci
isoelectric focusing or two-dimensional gel electropho- in the MHC, HLA-B, and HLA-DRB. HLA-A, HLA-B,
resis methods. In addition to clinical applications, these HLA-C, and HLA-DRB are all considered important for
typing methods were also applied to forensic identi- successful transplantation outcomes.26 For this reason,
fication. Protein methods are limited by the demands the number of alleles in these particular loci has risen
of the methodology and the ability to distinguish only significantly compared with the number of serologically
those proteins that have different net charges. Mass defined polymorphisms.
spectrometry has also been applied to the evaluation Whole-blood patient specimens collected in ethylene-
of HLA peptides for tissue and organ transplantation diaminetetraacetic acid (EDTA) anticoagulant are used
(immunopeptidomics).24,25 for DNA-based typing. Cell lines of known HLA type
are used for reference samples. Standards and quality An assay, for example, using 30 probes required
assurance for DNA-based assays have been established approximately 70 μl of PCR product. The amplicons
by ASHI (http://www.ashi-hla.org). DNA isolation can were denatured by addition of NaOH and spotted onto
be performed from white blood cell preparations (buffy a membrane in 1- to 2-μl volumes. Spotting was done
coat) or from isolated nuclei treated with proteinase K. manually with a multichannel pipet or by a vacuum
manifold with a 96-well plate format. The spotted DNA
Sequence-Specific Oligonucleotide Probe was dried, then permanently attached to the membrane
Hybridization by ultraviolet (UV) cross-linking (exposure to UV light)
or baking. Separate membranes were produced for each
Hybridization of a labeled probe to immobilized ampl-
probe to be used. Every membrane included reference
icons of the HLA genes (dot blot) was one of the first
amplicons complementary (positive control) and non-
methods that utilized polymerase chain reaction (PCR)–
complementary (negative control) to all probes in the
amplified DNA for HLA typing. For this procedure
assay. Spotting consistency was checked using a con-
(Fig. 14.9), the HLA region under investigation is
sensus probe that will hybridize to all specimens on a
amplified by PCR using primers flanking the polymor-
membrane.
phic sequences. Because the majority of polymorphic
The probes used in this type of assay were short
sequences are located in exon 2 of the class II genes and
(19 to 20 bases), single-stranded DNA chains (oligonu-
exons 2 and 3 of the class I genes, primers are designed
cleotides) designed to hybridize to specific HLA alleles.
to target these regions.
The oligonucleotides were labeled, that is, covalently
attached to biotin or digoxygenin. Probe sequences
Specimen 1 (Type A*0203) Specimen 2 (Type A*0501) were based on sequence alignments of HLA polymor-
phic regions, aligned so that the polymorphic nucleotide
…TAGCGAT… …TAGAGAT… was in the middle of the probe sequence. Hybridization
…ATCGCTA… …ATCTCTA… conditions depended on the optimal hydrogen bonding
of probe complementary to a test sequence in compar-
Amplify, denature, ison with another sequence, differing from (not com-
bind to membrane plementary to) the probe by at least one base. In the
assay, spots of immobilized specimens bound to specific
probes gave a positive colorimetric or chemiluminescent
signal from the labeled probe. Panels of probes defined
specific alleles according to which probe bound the
Specimen 1 Specimen 2 immobilized amplified DNA under investigation. The
number of probes used depended on the design of the
assay. For example, an intermediate resolution assay of
Probe with allele-specific probes the HLA-DRB locus might have taken 30 to 60 probes.
Studies achieved high-resolution identification of the
…TAGCGAT… (A*02) …TAGAGAT… (A*05) majority of HLA-A, HLA-B, and HLA-C alleles using
67 HLA-A, 99 HLA-B, and 57 HLA-C probes and
intermediate resolution with 39 HLA-A and 59 HLA-B
alleles.27
Specimen 1 Specimen 2 Specimen 1 Specimen 2 Sequence-specific oligonucleotide probe (SSOP)
was also performed in a reverse dot-blot configuration
FIGURE 14.9 The principle of the SSOP assay is shown.
An HLA gene region is amplified from specimen DNA using in which the allele-specific probes were immobilized on
generic primers (top). The amplicons are immobilized on a the membrane (Fig. 14.10). In this method, the speci-
membrane and probed with labeled sequences complementary men DNA was labeled by PCR amplification using
to specific alleles. Signal from the bound probe will indicate primers covalently attached to biotin or digoxygenin at
the allele of the immobilized DNA. the 5′ end. In contrast to the SSOP described previously
where amplicons from each specimen were spotted on HLA-C class I and HLA-DRB1, DRB3, DRB4, DRB5,
multiple membranes, each specimen was tested for mul- and DQB1 antigens.
tiple alleles on a single membrane. Therefore, instead of SSOP is considered low to intermediate resolution,
having a separate membrane of multiple specimens for depending on the number and types of probes used in
each probe, a separate membrane of multiple probes was the assay. Because some probes have multiple specific-
required for each specimen. The reverse dot-blot strat- ities, hybridization panels can be complex. Computer
egy is now applied to bead array systems where fluo- programs may be used for accurate interpretation of
rescently distinct beads carry the oligonucleotide probes. SSOP results.
This system is used for typing of HLA-A, HLA-B, and
Sequence-Specific PCR
A faster method of sequence-based typing is the use of
sequence-specific primers that will amplify only specific
alleles (Fig. 14.11). As previously noted, the 3′ end of a
PCR primer must be complementary to the template for
recognition by DNA polymerase. By designing primers
Color that end on the polymorphic bases, successful generation
signal of a PCR product will occur only if the test sequence
has the polymorphic allele complementary to the primer.
Amplicon Detection of the PCR product is used to indicate spe-
Probe cific alleles. Sequence-specific PCR (SSP-PCR) is faster
and easier than SSOP in that no probes or labeling steps
are required, and the results of SSP-PCR are determined
FIGURE 14.10 In reverse dot-blot SSOP, the probe is immo- directly by agarose gel electrophoresis.
bilized on the membrane. Patient DNA is amplified using For SSP-PCR, isolated DNA is amplified using sets
primers covalently bound to biotin or digoxygenin at the
of primers designed to specifically amplify a panel of
5+ end. The amplicons are then hybridized to panels of probes
immobilized on a membrane (top). If the sequence of the ampl-
alleles. Reactions are set up in a 96-well plate format,
icon matches and hybridizes to that of the probe, a secondary with different allele- or sequence-specific primer sets in
reaction with enzyme-conjugated avidin or antidigoxygenin each well. Each PCR reaction mix contains sequence-
will produce a color or light signal when exposed to substrate. specific primers and amplification control primers in
If the sequence of the amplicon differs from that of the probe, a multiplex format. The amplification control primers
no signal is generated (bottom right). should yield a product for every specimen (except the
TCATGA…
Amplification controls
TGACTTGCATCGTGCATCT AGCTAGCTACAGTACTACATC
ACTGAACGTAGCACGTAGA TCGATCGATGTCATGATGTAG Allele-specific product
…CTTGCAT
Amplification
TCATGA…
TGACTTGCATCGTGCATCT AGCTAGCTACCGTACTACATC
ACTGAACGTAGCACGTAGA TCGATCGATGGCATGATGTAG
…CTTGCAT
No amplification
FIGURE 14.11 Sequence-specific PCR relies on the requirement of complementarity between the 3′ base of the primer and the
template. The sequence-specific primer ending in AGTACT will be extended only from a template carrying the polymorphism
shown.
FIGURE 14.12 Results of an SSP-PCR of

95 primer sets detected by agarose gel electro-
phoresis show the presence of specific alleles
as allele-specific PCR products. An amplifica- A
tion control (top band in each well) is included
with each reaction to avoid false-negative A
results due to amplification failure. Contami-
nation is monitored by a reagent blank. (Photo
courtesy of Christin Braun, Rush University Medical
Center.)
negative control). The sequence-specific primers should

only yield a product if the specimen has the allele com- Exon 2 Exon 3
plementary to (matching) the allele-specific primer
sequence. The amplification primers are designed to
yield a PCR product of a size distinct from the product of FIGURE 14.13 Example of primer placement for amplifica-
the allele-specific primers. The two amplicons can then tion and Sanger sequence analysis. PCR primers (outer large
be resolved by agarose gel electrophoresis. An illustra- arrows) are used to amplify the region of interest. The PCR
product is then sequenced using four different primers (inner
tion of the results expected from SSP-PCR is shown in
small arrows) in separate sequencing reactions.
Figure 14.12. Specimens will yield two PCR products
(amplification control and allele-specific product) only
from those wells containing primers matching the spec-
true for any DNA test, no less for discovery and identi-
imen HLA allele. Wells containing primers that do not
fication of HLA types. Classical sequence-based typing
match the patient’s HLA allele will have a band only
(SBT) involves amplification of HLA class I exons 2,
from the amplification control.
3, and 4 and class II exons 2 and 3 (Fig. 14.13). The
SSP-PCR is frequently performed on PCR plates
larger amplicons are purified and added to a sequencing
preloaded with reaction mixes (typing trays) containing
reaction mix for sequencing with the inner sequencing
primers specific for class I and class II DRB and DRQ
primers (Fig. 14.14). Following gel or capillary gel elec-
genes. Specimen DNA is introduced into the individual
trophoresis, the fragment patterns or electropherograms
wells, and the plates are placed in the thermal cycler.
of the DNA ladders are examined for specific polymor-
Plate maps of allele-specific primers are provided for
phisms (matches to stored sequence databases of known
interpretation of the HLA-type. SSP-PCR has become
alleles).
a commonly used method for testing potential donors
There are some technical challenges to sequence-
before transplant.
based typing, such as secondary structure and other arti-
facts that alter the mobility of fragments and complicate
Sequence-Based Typing
interpretation. Furthermore, several events can occur
The most definitive way to analyze DNA at the nucleo- that will compromise sequence quality. Manufacturers
tide sequence level is by direct DNA sequencing. This is of sequencing reagents supply ways to correct most
Isolate DNA of these problems. As with any sequencing assay, it is

useful to sequence both strands of the test DNA.
Because HLA haplotypes are almost always het-
erozygous, Sanger sequencing results often yield a
heterozygous pattern at the site of the polymorphism
PCR
(Fig. 14.15). Phasing or placement of alleles on the
same (maternal or paternal) chromosome in diploid
organisms is difficult in Sanger typing. Also, ambiguous
allele assignments revealed in rare alleles found by high-
resolution sequencing require additional testing and
delay results. Ambiguities are produced by a lack of
sequencing of all polymorphic positions and/or lack of
phasing. Therefore, exact high-resolution HLA typing is
increasingly difficult with the growing number of HLA
Clean amplicons alleles not easily distinguished from one another. Com-
mercial systems including PCR and sequencing reaction
mixes and software programs for interpretation of results
have been designed to address these issues.
Sequence amplicons
Next-generation sequencing (NGS) offers the advan-
tages of expanding the regions to be sequenced in
phase while lowering ambiguities. NGS HLA panels
cover the entire HLA-A, HLA-B, and HLA-C genes
for class I and the entire HLA-DQB1, HLA-DQA1, and
FIGURE 14.14 For Sanger sequence–based typing, HLA HLA-DPA1 genes and exon 2 of the HLA-DPB1 for
regions from patient DNA are amplified using the outer class II. These regions are covered by long-range PCR,
primers. The PCR products are then purified from unused PCR which allows accurate phasing of heterozygous alleles.
reaction components by alcohol precipitation or column or gel
Two technologies account for most HLA sequencing by
purification methods. The amplicons are then sequenced using
the inner sequencing primers to detect polymorphisms.
FIGURE 14.15 For a sequence-based

typing result for the HLA-A gene, soft-
ware is designed to analyze the sequence of
patient DNA, compare it with the consensus
sequence, and identify the specific alleles
by sequence polymorphisms. (Sequence cour-
tesy of Christin Braun, Rush University Medical
Center.)
Class II Class I
DP DQ DR B C A
B1 A1 B1 A1 B1 B3-5 A1
Adapter Adapter
Bar code
Binds flow cell or bead
FIGURE 14.16 Next-generation sequencing libraries are produced from MHC regions selected by long-range PCR (double
arrows). These 7- to 10-kb fragments are fragmented or amplified for generation of shorter fragments. The fragments are ligated to
adapters and then tagged by PCR using primers tailed with bar codes for patient identification and sequencing primer-binding sites.
In dye terminator sequencing, sequencing reactions are performed from polonies immobilized on a sequencing chip (flow cell). For
ion conductance, sequencing reactions are performed on beads in wells of the sequencing chip.
NGS, reversible dye terminator sequencing28 and ion is then used to assign haplotypes by comparison of the
conductance.29 alleles to consensus sequences in the IMGT/HLA data-
For NGS typing, HLA genes are first amplified by base of alleles.
long-range PCR (Fig. 14.16). The resulting PCR prod-
ucts range from 4 to 10 kb in size. The sequencing library
Other DNA-Based Methods
is prepared from the amplified fragments by enzymatic
fragmentation to products less than 1 kb in size and Almost any method that can determine DNA sequence
addition of secondary primer-binding sites. These sites or detect specific sequences or sequence differences can
are used to amplify the fragments with primers carrying be applied to DNA-based HLA typing. There are several
patient-specific sequences (bar codes) and recognition variations on SSOP, SSP, and SBT, such as the nested
sites for immobilized sequencing primers (Fig. 14.16). PCR-SSP30 proposed for high-sensitivity HLA typing.
The bar codes allow sequencing of multiple patient Another example is allele-specific nested PCR-SSP,
samples simultaneously, with their sequences assigned which has been applied to subtyping of highly polymor-
to each patient by the bases in the bar code. Depending phic alleles.
on the type of sequencer, the sequencing primers may be Heteroduplex (HD) analysis was used for assessment
attached to beads or directly to a chip or flow cell. of compatible bone marrow donors at the HLA-DR and
After amplification, sequencing proceeds directly HLA-DP loci.31 Reference strand conformation poly-
on the flow cell. Sequencing by ion conductance is morphism was a variation on the standard HD analy-
performed in wells on the chip, each accommodat- sis; sample amplicons were mixed with fluorescently
ing a bead on which each library fragment has been labeled reference DNA of known allele sequence before
amplified. Library concentration and sequence quality denaturation and renaturation to form HD. The homodu-
are monitored in the course of sequencing. As with plexes and HD formed between the specimen amplicons
Sanger-based NGS, instrument software can analyze and the reference strand were then resolved by capillary
the sequence data. Specialized HLA analysis software gel electrophoresis.
Single-strand conformation polymorphism (SSCP)

was also applied to HLA-A, DR, DQ, and DP typing and TABLE 14.6 Resolution of HLA Typing Methods
subtyping, sometimes coupled with allele-specific PCR.
High-performance liquid chromatography (HPLC) was Intermediate
Low Resolution Resolution High Resolution
proposed for HLA typing as well. Conformation analy-
ses, such as HD, SSCP, and HPLC, however, are limited CDC (serology) PCR-SSP PCR-SSP
by the complexity of the raw data and the strict demands
on reactions and electrophoresis conditions; they are not PCR-SSP PCR-SSOP PCR-SSOP
routinely used in the clinical laboratory. PCR-SSOP PCR-RFLP SSP-PCR + PCR-RFLP
Other DNA-based typing approaches include array
technology32 and pyrosequencing.33 No one method is SSOP-PCR + SSP-PCR
without disadvantages with respect to technical demands, SBT
cost, or time consumption. To date, SSP, SSOP, Sanger
sequencing, and NGS are the DNA-based methods used
in most clinical laboratories.
the typing resolution of DNA-based tests. Serological
testing can be used to clarify or confirm the phenotype
Combining Typing Results
of alleles detectable by DNA-based methods. The reso-
At the DNA level, HLA polymorphisms differ from one lution of results from various methods, therefore, reflects
another by as little as a single nucleotide base. Serolog- a range of resolution levels (Table 14.6). The choice of
ical typing does not always distinguish subtle genetic method will depend on the demand for high- or low-
differences between types, and also requires the proper resolution typing.
specimen. A specimen consisting of mostly T cells, for
example, from a patient treated with chemotherapy will
HLA Test Discrepancies
not provide B cells (which carry class II antigens) for
testing of class II haplotypes. In contrast, DNA-based HLA typing may produce discrepant results, especially
typing is not limited by specimen type because all cells if different methods are used to assess the same speci-
have the same HLA haplotype at the DNA level, regard- men. The most common discrepancies are those between
less of whether the cell type expresses the antigens. serology and molecular testing results.
Furthermore, synonymous DNA changes and polymor- DNA sequence changes do not always affect protein
phisms outside of the protein-coding regions may not epitopes. A serology type may represent several alleles
alter antigenicity at the protein level. at the DNA level. Also, a serology type may look homo-
For DNA-based methods, the design of molecular zygous (match to only one antibody) where the DNA
methods (primer and probe selection) determines their alleles are heterozygous, the second allele not being rec-
level of resolution. SSOP and SSP methods require spe- ognized by serology. For example, a serology type of A2
cific primers and/or probes for each particular HLA type. is determined to be A*02, A*74 at the DNA level.
Only those HLA types included in a given probe or primer Discrepancies also arise when HLA types assigned to
set, therefore, will be identified. Sanger sequence–based parent alleles based on DNA sequence homology differ
typing will only identify alleles included in the ampli- from serology results that detect the same allele as a
fied regions that are sequenced. NGS provides almost new antigen. For example, a DNA allele of B*40:05 is
complete coverage of HLA loci with better accuracy; detected as a new antigen by serology and named B50.
however, this technology has yet to be implemented in Similarly, split alleles (subtypes of serologically defined
many laboratories. antigens) can differ between DNA and serology typing
Results from serological and DNA-based methods due to the cross reactivity of antibodies used to define
can be combined to improve resolution and further the HLA antigens.
define HLA types. Sequential use of SSP-PCR and The identification of new alleles can result in ambi-
PCR-RFLP or SSOP-PCR and SSP-PCR increases guities and discrepant retyping results based on the
recognition of new alleles that were not defined at the transplants can be carried out by molecular methods
time of an initial typing. These discrepancies may be such as SSP-PCR, sequencing, or even genome-wide
difficult to resolve, especially if the original typing data correlations of mHag sequences with single-nucleotide
are not accessible. Sequence ambiguities can be resolved polymorphisms from the HapMap project.41,42
by NGS, if available to the laboratory.
Nonconventional MHC Antigens
Coordination of HLA Test Methods Located within the MHC locus are the MHC class
The choice of the appropriate method and resolution of I–related MICA and MICB genes. Three pseudogene
HLA testing is influenced by the type of transplant. For fragments, MICC, MICD, and MICE, are also found
solid organ transplants, antibody screening and cross- within the class I region. The products of the MICA and
matching of recipient serum against donor antigens are MICB genes, along with those of the retinoic acid early
routinely performed, although not always before trans- transcript (RAET) gene cluster, located on the long arm
plantation. Pretransplant HLA typing is often deter- of chromosome 6 (6p21.3), bind to the receptor NKG2D
mined for kidney and pancreas transplants because the on natural killer (NK) cells (killer cell lectin-like recep-
extent of HLA matching is directly proportional to the tor, subfamily K, number 1 or KLRK1). A soluble
time of survival of the donor organ. Given the circum- isoform of MICA (sMICA) has the potential to suppress
stances under which heart, lung, and liver transplants NKG2D-mediated host innate immunity by promoting
are performed, testing is frequently performed after the degradation of the receptor-ligand complex. These gene
transplant. HLA typing for solid organs is usually at the products participate in immune reactions against abnor-
low-resolution serology level, although for heart and mal cells such as tumor cells through control of NK cells
lung transplants, as with kidney transplants, studies have and cytotoxic T lymphocytes (CTLs) expressing the
shown that matching HLA types are beneficial for organ γδ T-cell receptor. Virus- or bacteria-infected cells may
survival.34 For stem cell and bone marrow transplants, also be recognized and eliminated in part by this system.
typing to high resolution (specific alleles) is preferred The MICA and MICB genes are highly polymor-
in order to decrease the risk of rejection and to avoid phic. Approximately 107 MICA and 42 MICB alleles
GVHD.35 NGS has provided typing up to 4-digit allelic have been reported. In contrast to the MHC class I
levels for this purpose.36 alleles, polymorphisms in the MIC genes are distributed
throughout the coding regions, with no hypervariable
regions. Anti-MIC antibodies have been detected after
organ transplantation, similar to anti-HLA alloantibod-
ADDITIONAL RECOGNITION FACTORS ies, supporting a role for these gene products in organ
rejection.43
Minor Histocompatibility Antigens
Any donor protein that can be recognized as nonself
Killer Cell Immunoglobulin-Like Receptors
by the recipient immune system can potentially affect
engraftment. Proteins outside the MHC that influence NK cells and some memory T cells express killer cell
graft failure are called minor histocompatibility anti- immunoglobulin-like (KIR) proteins. The effect of
gens (mHags). These antigens are the suspected cause these proteins was first observed as “hybrid resistance”
of GVHD and graft rejection in MHC-identical trans- in mice.44 In these experiments, mice with compro-
plants.37,38 Conversely, mHags may promote graft- mised immune systems were still capable of rejecting
versus-tumor (GVT) effects as well.38 Identification of grafts from unrelated mice. That is, graft rejection still
tissue-specific mHags may allow inhibition of GVHD occurred, even in the absence of a functional immune
while increasing GVT activity. system. The KIR proteins have been proposed as one
The H-Y antigen was the first characterized mHag.39 source of nonself recognition outside of the MHC. The
Molecular methods have led to the characterization of KIR proteins interact with HLA antigens, specifically
additional mHags.40 Evaluation of mHags in stem cell recognizing HLA-A, HLA-B, and HLA-C (class I)
Centromeric Telomeric
2DL2 2DS3 3DP1 3DS1 2DL5A

3DL3 2DS2 2DL5B 2DP1 2DL1 2DL4 2DS1 3DL2
2DL3 2DS5 3DP1v 3DL1 2DL5B
2DS3 2DS4
2DS5
FIGURE 14.17 The KIR gene cluster includes a centromeric and a telomeric fragment. Gene content varies from one person to
another. A KIR haplotype can contain from 8 to 14 genes in different combinations and gene orders. For example, a haplotype
may have a 2DL2 or 2DL3 gene between the 2DS2 and 2DL5B genes, or the order of 2DS1 and 2DS4 can be 2DS1–2DS4 or
2DS4–2DS1.
molecules. KIR proteins are also expressed on myelo-

monocytic lineage cells (leukocyte immunoglobulin-like TABLE 14.7 KIR and HLA Gene Interactions
receptor) and other leukocytes (leukocyte-associated
immunoglobulin-like receptor). A cluster of genes KIR HLA Specificities
coding for these receptor proteins has been found on
2DL1, Cw*02, *03:10, *03:04, *03:05, *03:06, *03:07,
chromosome 19q13.4, the leukocyte receptor cluster 2DS1 *03:15, *07:07, *07:09, *12:04, *12:05, *15 (except
(Fig. 14.17). (2DS4) *15:07), *16:02, *17, *18
Recipient KIR may participate in graft rejection
2DL2, Cw*01, *03 (except *03:07), *03:10, *03:15, *07
and donor KIR in GVHD.45 Treatment for GVHD with
2DL3 (except *07:07, *07:09), *07:08, *12 (except *12:04,
cyclophosphamide after transplant may also inhibit the (2DS2) *12:05), *13, *14, *15:07, *16 (except *16:02)
GVT effect of NK cells.46 Specific interactions between
KIR and HLA genes are listed in Table 14.7. Just as with 2DL5 Unknown
mHags, assessment of polymorphisms in KIR may be 3DL1 Bw4
added to donor selection criteria in stem cell and bone
marrow transplants, especially with unrelated donors. 3DL2 A3, A11
In contrast to HLA typing, testing for KIR is aimed at 3DL3 Unknown
finding donors and recipients who do not match. KIR
alleles are typed by SSO using bead array technology 2DS3 Unknown
and by NGS.47 2DS5 Unknown
3DS1 Bw4
MHC DISEASE ASSOCIATION
Genetic diseases caused by single-gene disorders obey
Mendelian laws. Their phenotypes are either dominant analysis yields results in terms of predisposition, proba-
or recessive and are inherited in a predictable manner, bility, and risk of disease.
as illustrated in pedigrees. Most diseases, however, Autoimmune diseases, which affect 4% of the pop-
are not caused by a single genetic lesion and therefore ulation, fall in this category. At least one of the genetic
have complex segregation patterns. Multiple genes, epi- factors involved in autoimmunity is linked to the MHC
genetics, and environmental factors combine to bring because autoimmune diseases have MHC associa-
about these disease states. For diseases such as diabe- tions.48,49 Rheumatoid arthritis, multiple sclerosis, diabe-
tes, high blood pressure, and certain cancers, genetic tes mellitus type 1, and systemic lupus erythematosus
are associated with particular HLA haplotypes. Deter-

mination of a disease-associated HLA haplotype aids in TABLE 14.8 Summary of Testing
diagnosis or prediction of disease predisposition. for Pre- and Post-Transplant Evaluation
The presence of a haplotype associated with disease
is not diagnostic on its own, however. An example is Purpose Test Design Methods
the HLA-B27 type found in all cases of ankylosing Determine HLA Determine HLA Serology
spondylitis. HLA-B27 is also the most frequently found type types with standard
HLA-B allele. Many people, therefore, with the B27 references or by DNA
allele do not have this condition. Other associations, sequence
such as narcolepsy being strongly associated with HLA- DNA-based
DQB1*06:02, may arise from involvement of presenta- typing
tion of an autoantigen to CD4+ T cells in the context
of specific HLA types and subsequent autoimmunity.50 Determine Serum screening CDC
serum antibody against known HLA
The laboratory is usually asked to perform HLA typing status antibodies
in persons showing disease symptoms or family his-
tories of such disease to aid or confirm the diagnostic ELISA
decisions.
Flow cytometry
Normal states are also controlled by multiple genetic
and environmental factors. The genes for olfactory Crossmatching Compare serum of CDC
sensation, histone genes, genes encoding transcription recipient with that of
prospective donors
factors, and the butyrophilin (a constituent of milk in
mammals) gene cluster are found in the MHC. The MHC ELISA
may play a role not only in the predisposition to disease
but also in protection from disease, including some Flow cytometry
infectious diseases, notably HIV.51 In this role, certain Determine Alloreactive T-cell MLC
HLA types are considered “protective.” It has been sug- T-cell-mediated characterization and
gested that HLA type be considered in antiviral therapy cytotoxicity quantitation
trials.52 between donor
and recipient
Cytokine
SUMMARY OF LABORATORY TESTING production
Standard HLA testing includes a range of methods and

test designs (Table 14.8). Determining donor and recip-
ient compatibility is the primary goal of pretransplant Advanced Concepts
testing, especially for bone marrow transplant from
unrelated donors. The extent of HLA matching will HLA types are also associated with the absence
increase the prospect of successful transplantation with of inherited disease. Protection from genetic dis-
minimal GVHD. For example, the NMDP requests HLA eases is naturally enhanced by “hybrid vigor” or
typing results for HLA-A, HLA-B, and HLA-C class I avoidance of inbreeding. The MHC may bring
antigens and DRB1, DQB1, and DPB1 class II antigens about such natural protection by control of mating
for prospective bone marrow donors. Advances in SBT selection to ensure genetic mixing. For example,
using NGS have increased the resolution of typing and studies with mice have shown that olfactory sen-
limited the number of ambiguities that formerly had sation (sense of smell) may play a role in mate
to be resolved by other methods, thus shortening the selection. In these studies, female mice were able
reporting time for typing.
to distinguish male mice with H-2 alleles different Case Study 14.1
from their own.53 This supports the idea of selec-
tive pressure to mate with HLA-dissimilar part- A 50-year-old man complained of digestive dis-
ners to increase genetic diversity. Another study orders and what he presumed was an allergy to
in humans showed that women found the scent of several foods. He consulted his physician, who
T-shirts worn by some male subjects pleasant and collected blood samples for laboratory testing.
others unpleasant. The odors detected as pleasant The man was in a high-risk group for certain
were from HLA-dissimilar males. The women diseases, notably celiac disease, which would
were apparently able to distinguish HLA types dif- produce symptoms similar to those experienced
ferent from their own through the sweat odors of by this patient. A specimen was sent to the lab-
male test subjects.54 oratory to test HLA-DQA and HLA-DQB alleles.
Almost all (95%) people with celiac disease
have the DQA1*05:01 and DQB1*02:01 alleles,
compared with 20% of the general population. A
Combinations of molecular tests, serum screening, second predisposing heterodimer, DQ8, is encoded
and crossmatching further define acceptable HLA mis- by the DQA1*03:01 and the DQB1*03:02 alleles.
matches, and the results are used to prevent hyperacute Most of the 5% of celiac patients who are nega-
(almost immediate) rejection in organ transplants. Iden- tive for the DQ2 alleles display the DQ8 alleles.
tification of HLA types associated with disease also Serological results to detect the predisposing anti-
provides important information, especially for disease gens, however, were equivocal. An SBT test was
caused by multiple genetic and environmental factors. performed. The indicated alleles were detected by
Finally, MLC, although not generally part of routine sequence analysis. The sequence results showed
clinical testing, can predict cellular factors involved in the following alleles at DQA1 and DQB1 loci:
rejection.
Laboratory results are key for the selection of com- DQA1 * 05 : 01 DQB1 * 02 : 01, DQB1 * 04 : 01
patible donors, post-transplant evaluation, selection of
optimal treatment strategy, and evaluation of genetic QUESTION: Is it possible that this man has celiac
disease predisposition. Molecular analysis has signifi- disease based on his HLA haplotype?
cantly increased the ease and ability to detect subtle dif-
ferences in the MHC and associated regions. Successful
organ transplantation will increase as databases grow
and instrumentation, automation, and bioinformatics
advance. Nucleic acid analysis has contributed greatly to
more definitive analysis in this area of laboratory testing.
Case Study 14.2

A 43-year-old man consulted his physician about a rearrangement. The patient was initially treated with
lump on his neck and frequent night sweats. A biopsy standard chemotherapy, but the tumor returned before
of the mass in his neck was sent to the pathology the therapeutic program was completed. The tumor
department for analysis. An abnormally large popu- persisted through a second treatment with stronger
lation of CD20-positive lymphocytes was observed chemotherapy plus local irradiation. Nonmyeloab-
by morphological examination. Flow-cytometry lative bone marrow transplant was prescribed. To
tests detected a monoclonal B-cell population with find a compatible donor, the man’s HLA type and
coexpression of CD10/CD19 and CD5. This popu- the types of five potential donors were compared.
lation was 88% kappa and 7% lambda. The results HLA-A and HLA-B types were assessed by serology,
were confirmed by the observation of a monoclonal and HLA-DR type was determined by SSP-PCR and
immunoglobulin heavy-chain gene rearrangement SSOP. The typing results are shown in the accompa-
that was also monoclonal for kappa light-chain gene nying table.
Recipient Donor 1 Donor 2 Donor 3 Donor 4 Donor 5
A*03:02 A*66:01/04 A*26:01 A*03:02 A*31;03/04 A*26;10
A*26:10 A*03:02 A*43:01 A*03;09 A*0309
B*39:06 B*07:11 B*15:28 B*38:01 B*27;02 B*35;08
B*53:07 B*57:01 B*39:19 B*39:01 B*3501
DRB1*08:01 DRB1*07:01 DRB1*13:17 DRB1*08:09 DRB1*07;01 DRB1*13;17
DRB1*13:17 DRB1*04:22 DRB1*11:17 DRB1*07;01
QUESTIONS: 2. Using the donor bone marrow that matches most

1. Which of the five donors is the best match for this closely, is there more of a chance of graft rejection
patient? Which mismatches are acceptable? or GVHD?
Case Study 14.3

A 19-year-old woman reported to a local clinic with was compromised, and she would eventually suffer
painful swelling in her face. Routine tests revealed kidney failure. With an alternative of lifelong dial-
dangerously high blood pressure that warranted hos- ysis, a kidney transplant was recommended. Class I
pitalization. Further tests were performed, which led PRAs were assessed at 0% PRA for class I. An addi-
to a diagnosis of systemic lupus erythematosus. Due tional screen for class II PRA was performed by flow
to complications of this disease, her kidney function cytometry.

Case Study 14.3 (Continued)

50
M1 M2
40
Counts
30
20
10
0
0 200 400 600 800 1000
Sera
50
M1 M2
40
Counts
30
20
10
0
0 200 400 600 800 1000
SSP-PCR results from an HLA-A typing tray for the daughter.
Sera
Alleles were identified in lanes 7, 19, and 22.
50
M1 M2
40 SSP-PCR results for HLA-A, HLA-B, and
HLA-DR and their serological equivalents are shown
Counts
30
in the accompanying table.
20
Crossmatching of the daughter ’s serum and the
10
mother ’s cells was performed by cytotoxicity and
0 flow cytometry. Both test results were negative.
0 200 400 600 800 1000
Sera QUESTIONS:
Flow-cytometry analysis of HLA class II antigens in recipi- 1. Is the mother a good match for the daughter?
ent serum. Top = negative control; middle = positive control;
bottom = patient serum.
2. Based on the antibody and crossmatching studies,
what is the risk of rejection? Before performing the
Class I and class II HLA typing was performed HLA studies, how many of the daughter's antigens
by SSP-PCR. An autocytotoxic crossmatch was also would be expected to match those of her mother?
performed, revealing T-cell- and B-cell-positive anti-
bodies, probably related to the lupus. The young Daughter Mother
woman’s mother volunteered to donate a kidney to
her daughter. Mother and daughter had matching SSP-PCR Serology SSP-PCR Serology
blood group antigens and HLA-DR antigens, which A*24:19, A24(9), A*11:04, A11, A24(9)
are the most critical for a successful organ transplant. A*3401 34(10) A*24:19
The results from the typing tray are shown in the fol-
B*08:02, B8, B22 B*08:04, B8, B22
lowing figure:
B*56:03 B*56:03
DRB1*04:22, DR4, DRB1*04:22, DR4,

DRB1*14:11 DR14(6) DRB1*13:17 DR13(6)
8. A CDC assay yields an 8 score for sera with the

STUDY QUESTIONS following specificities: A2, A28 and A2, A28, B7,
and a 1 score for serum with an A2 specificity.
1. Which of the following is a high-resolution HLA What is the HLA-A type?
typing result?
9. HLA-DRB1*15:01 differs from
a. B27 DRB1*01:01 by a G to C base change. If
b. A*02:02–02:09 the sequence surrounding the base change
c. A*02:12 is …GGGTGCGGTTGCTGGAAAGAT…
d. A*26:01/A*26:05/A*26:01/A*26:15 (DRB1*01:01) or …
GGGTGCGGTTCCTGGAAAGAT…
2. Which of the following is a likely haplotype from (DRB1*15:01), which of the following would
parents with A25,Cw10,B27/A23,Cw5,B27 and be the 3′ end of a sequence-specific primer for
A17,Cw4,B10/A9,Cw7,B12 haplotypes? detection of DRB1*15:01?
a. A25,Cw10,B27 a. …ATCTTTCCAGGAACCC
b. A25,Cw5,B27 b. …ATCTTTCCAGCAACCC
c. A23,Cw4,B12 c. …ATCTTTCCAGC
d. A17,Cw4,B27 d. …ATCTTTCCAGG
3. Upon microscopic examination, over 90% 10. The results of an SSP-PCR reaction are the
of cells are translucent after a CDC assay. following: lane 1, one band; lane 2, two
How are these results scored according to the bands; lane 3, no bands. If the test includes an
ASHI rules? amplification control multiplexed with the allele-
specific primers, what is the interpretation for each
4. An HLA-A allele is a CTC to CTT (leu → leu) lane?
change at the DNA level. How is this allele
written?
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Chapter 15
Quality Assurance and Quality Control
in the Molecular Laboratory
Outline Objectives
SPECIMEN HANDLING 15.1 Describe proper specimen accession for molecular
Collection Tubes for Molecular Testing testing.
Precautions 15.2 Describe the optimal conditions for holding and
Holding and Storage Requirements storage of specimens and nucleic acid.
TEST PERFORMANCE 15.3 Explain the basic components of molecular test
Next-Generation Sequencing performance, including quality assurance and
Calibrators and Method Calibration controls.
Controls 15.4 Discuss instrument maintenance, repair, and
QUALITY CONTROL calibration, particularly for instruments used in
QUALITY ASSURANCE molecular analysis.
INSTRUMENT MAINTENANCE 15.5 Describe recommendations for the preparation
Instrument Calibration and use of reagents in the molecular laboratory.
REAGENTS 15.6 Explain documentation and reporting of results,
Reagent Categories including gene sequencing results.
Chemical Safety
Reagent Storage
Reagent Labeling
PROFICIENCY TESTING
DOCUMENTATION OF TEST RESULTS
Gene Nomenclature
Gene Sequencing Results
REPORTING RESULTS
446
Chapter 15 • Quality Assurance and Quality Control in the Molecular Laboratory 447
Congress passed the Clinical Laboratory Improve- of collection, reason for testing, and the contact informa-
ment Amendments (CLIA) in 1988 to establish quality tion (pager or telephone number) of the ordering physi-
testing standards to ensure consistent patient test results. cian. When required (for molecular genetics, forensics,
CLIA specifies quality standards for proficiency testing, or parentage testing), patient consent forms, ethnicity,
patient test management, quality control, personnel photo identification of the individuals tested, patient
qualifications, and quality assurance for laboratories label verification, and transfusion history or a pedigree
performing moderate- and/or high-complexity tests, may also be supplied with the test specimen. Forensic
including molecular testing. This chapter offers a brief specimens may require a documented chain of custody.
overview of laboratory standards applied to molecular Bar coding of this information expedites specimen acces-
diagnostic tests. sion. The laboratory should have written procedures for
documentation of specimen handling and accession.
Accession books or electronic records are used to record
SPECIMEN HANDLING the date of receipt, laboratory identifier, and pertinent
patient information associated with the accession. If a
Molecular tests, like any clinical laboratory tests, require specimen is unacceptable, the disposal or retention of
optimal specimen handling and processing for accurate the specimen is recorded in the patient report or labora-
and consistent test results. The success of a test proce- tory quality assurance records. If the specimen cannot be
dure is affected by the age, type, and condition of spec- tested, the ordering physician is notified.
imens. Therefore, specimen collection, transport, and If not processed immediately, specimens are main-
handling in the laboratory require careful attention. tained in secure areas with limited access under the
Preanalytical variables, both controllable and uncon- appropriate conditions for the analyte being tested
trollable, must be taken into account for proper inter- (Fig. 15.1). Care is taken to avoid contamination or
pretation of test results. Preanalytical error is the mixing if specimens are aliquoted. Slides of thin sections
consequence of erroneous or misleading results caused used for in situ hybridization procedures are retained for
by events that occur prior to sample analysis. To mini-
mize preanalytical error and maximize control of prean-
alytical variables, the Clinical and Laboratory Standards
Institute (CLSI, formerly known as the National Com-
mittee for Clinical Laboratory Standards) provides rec-
ommendations for the collection of specimens under
standardized conditions.
Each laboratory will have requirements for speci-
men handling, but general policies apply to all specimen
collection. A requisition or electronic test order should
accompany the specimen. The condition of the speci-
men and, if necessary, the chain of custody is reviewed
on receipt in the laboratory. If a specimen shows evi-
dence of tampering or is hemolyzed, degraded, clotted,
or otherwise compromised, the technologist must notify
the supervisor. No specimen is accepted without proper
labeling and identification on the specimen tube or con-
tainer (placed by the person who collected the speci-
men), nor is a specimen accepted if the labeling on the
specimen does not match that on the accompanying req-
uisition. In addition to relevant patient identification, the FIGURE 15.1 Biohazard stickers are required for cabinets,
test requisition includes the type of specimen material refrigerators, or freezers that contain potentially hazardous
(e.g., blood or bone marrow), ordered test, date and time reagents or patient specimens. See Color Plate 10.
10 years or 20 years for neoplastic or constitutional anal-

yses, respectively.
Molecular amplification methods have enabled lab-
oratory professionals to perform nucleic acid–based
testing on specimens with minimal cellular content,
such as buccal cell suspensions and cerebrospinal fluids.
These samples are centrifuged to collect the cells before
DNA or RNA is extracted. For routine specimens tested
by amplification methods, the entire specimen is often
not used. In this case, or if more than one test is to be
performed on the same specimen, cross-contamination
of specimens is carefully avoided. This can most likely
occur from pipetting carryover. Moreover, an aliquot FIGURE 15.2 Tissue is received in the laboratory in fresh
removed from a specimen is never returned to the orig- (left) or frozen (right) form. Fresh tissue may be supplied as a
inal tube or vessel. Molecular genetic tests may require specimen on gauze or another substrate or in saline. The small
dedicated specimens not shared with other tests. vial shown on the right contains tissue that was flash-frozen in
isopentane. The vial is held immersed in liquid nitrogen, in
Hemoglobin inhibits enzyme activity. Specimens
nitrogen vapors, or in a –70°C freezer.
received in the laboratory should therefore be inspected
for visual signs of hemolysis. Hemoglobin and coagu-
lants are removed effectively in most DNA and RNA tissue areas to be tested. For oncology testing, preanalyt-
isolation procedures; however, if white blood cell lysis ical review of the H&E slide by a pathologist is required
has also occurred, DNA or RNA yield will be reduced. to confirm adequacy and to designate what part of the
This could result in false-negative results in qualitative tissue sample will be tested. Tumor tissue samples are
testing or inaccurate measurements in quantitative anal- not always well defined or may be infiltrated with lym-
yses. Buffers and resins have been designed to seques- phocytes. Therefore, the pathologist may also estimate
ter anticoagulants or hemoglobin for more rapid nucleic the percentage of tumor cells in the area to be tested.
acid isolation without the inhibitory effects of these PCR and reverse transcription PCR (RT-PCR) ampli-
substances. fication are routinely performed on paraffin-embedded
Solid tissues are best analyzed from fresh or frozen tissue samples. Methods such as Southern or northern
samples (Fig. 15.2), especially for Southern blot or blot, requiring large fragments of DNA, are less likely
long-range polymerase chain reaction (PCR) methods to work consistently with fixed tissues.
that require relatively high-quality (long, intact) DNA.
Surgical specimens designated for molecular studies,
Collection Tubes for Molecular Testing
if not processed immediately, should be snap-frozen
in liquid nitrogen. This process preserves both nucleic Phlebotomy collection tubes are available with a number
acid and most gene-expression patterns that may change of different additives designed for various types of clini-
upon tissue storage. Snap freezing is routinely per- cal tests. A selection of collection tubes commonly used
formed in the surgical pathology laboratory because it is for molecular biology studies is listed in Table 15.1.
a common process for preserving tissue morphology for Some anticoagulants used in blood and bone marrow col-
microscopic examination. lection may adversely affect analytical results. Heparin
Fixed, paraffin-embedded tissues generally yield has been shown to inhibit enzymes used in molecular
lower-quality DNA and RNA, depending on the type analysis, such as reverse transcriptases and DNA poly-
of fixative used, the amount of time of exposure of the merases in vitro.1,2 The influence of this inhibition on
tissue to the fixative, and how the specimen was handled molecular analysis is commonly accepted; however,
prior to fixation. An advantage of fixed tissue is the use heparinized samples have been processed successfully
of adjacent slices of tissue that have been stained with in many laboratories, and acid citrate dextrose (ACD)
hematoxylin and eosin (H&E) to identify particular tubes are also used routinely for molecular tests.
TABLE 15.1 A Selection of Collection Tubes
Additive Color Nucleic Acid Testing
None Red Chemistry, serum,

viral antibody studies
Sodium heparin Green Immunology, virology

(freeze-dried) studies
Sodium heparin Brown Cytogenetic studies,

molecular studies
Tripotassium EDTA Lavender Virology, molecular

(7.5%–15% solution) biology studies
Acid citrate dextrose Yellow Molecular biology

solution studies
FIGURE 15.3 For molecular analysis, blood or bone marrow
specimens collected in EDTA (lavender-cap) or ACD (yellow-
cap) tubes are preferred. Heparin (green cap) is used for cyto-
The various experiences with heparin may reflect genetic tests. Immunoassays or mass-spectrometry methods
may be performed on serum collected in tubes without coagu-
levels of resistance of enzymes from different sources.
lant (red cap tubes). See Color Plate 11.
The assay design also has an effect. For instance, the
inhibition of DNA polymerases compromises amplifica-
tion of larger PCR products more than short ones. Due In addition to the standard collection tubes, special
to the possible effects of heparin, trisodium ethylene- collection tubes are designed particularly for stabiliza-
diaminetetraacetic acid (EDTA; lavender-top) or ACD tion of nucleic acids for molecular testing. These include
(yellow-top) tubes are recommended for most nucleic the plasma preparation tubes (Vacutainer PPT, Becton
acid assays involving enzymatic treatment of the sample Dickinson), which contain, in addition to the standard
nucleic acid (Fig. 15.3).3 High levels of disodium EDTA anticoagulants, a polymer gel that separates granulocytes
(royal blue–capped tubes used for trace element studies) and some lymphocytes from erythrocytes upon centrifu-
may also inhibit enzyme activity and should be avoided. gation. This type of tube is used for HIV and hepatitis
One of the advantages of signal amplification methods C virus (HCV) analysis. The PPT tube is also used for
such as bDNA technology is their decreased suscepti- separation of white cells for genetics, flow cytometry,
bility to the chemical effects of anticoagulants. When and engraftment testing. The tube stoppers have a black
a specimen is received in the laboratory, the anticoag- “tiger” pattern over the appropriate anticoagulant color
ulant is not usually noted in the accompanying doc- designation.
umentation; the technologist should be aware of the Specialty collection tubes are designed to stabilize
type of collection tube used, especially if a specimen is RNA. These tubes contain proprietary RNA stabiliza-
received in heparin or other additive that may affect the tion agents that maintain the integrity of the RNA from
test results. collection through isolation.4-6 Separated white blood
Tissue processing for molecular analysis requires cells from standard collection tubes can also be lysed in
caution to avoid contamination, especially where PCR phenol-containing solvents and the lysate stored at
or other amplification-based tests are to be performed. –70°C to stabilize RNA for several days.
Recommendations for tissue processing for molecu- Stabilization of transcripts has become increas-
lar diagnostic testing may be found in CLSI document ingly important with the increased use of methods
MM13A, “Collection, Transport, Preparation, and measuring RNA transcript levels. For example, quan-
Storage of Specimens for Molecular Methods; Approved titative RT-PCR analyses rely on transcript number to
Guideline.” monitor the level of tumor cells or microorganisms.
Serial analysis requires that the specimens received in

the laboratory at different times be handled as consis-
tently as possible. Immediate stabilization of the RNA
is important for consistent results. The laboratory pro-
cedure should include pre-analytical methods for spec-
imen preservation and storage conditions. There are a
variety of published methods for nucleic acid isolation;
some accompany reagent sets used for testing of par-
ticular analytes. Isolated RNA quantity and quality can
be measured where appropriate. Spectrophotometry is
usually used for this purpose; however, fluorometry and
gel electrophoresis are also used.
An alternative to standard collection tubes is the use
of impregnated paper matrices for nucleic acid preser-
vation and storage.7,8 Specimens stored in this way are
appropriate for amplification procedures (PCR) but not
for assays requiring larger amounts of nucleic acids.
FIGURE 15.4 Handling specimens with gloves is recom-
Precautions mended to protect the technologist and to protect the sample
nucleic acids from nucleases and other contaminants.
Standard precautions are recommended by the Centers
for Disease Control and Prevention for handling poten-
tially infectious specimens. All specimens are potentially
infectious, so they should all be handled with standard
precautions using proper personal protective equip-
ment (PPE) to prevent disease transmission. Transmis-
sion-based precautions including respirators are used
with airborne or contact-transmissible agents. Contact
precautions are designed for direct patient care where
there is the potential for direct exposure to infectious
agents on or from the patient. In general, standard pre-
cautions, including gloves and gowns as PPE, are used
by the molecular laboratory technologist who has no
direct contact with patients. Eye protection or masks are
required in cases where frozen tissue is being processed
or where spraying or splashing of a sample may occur.
Gloves are important, not only as part of stan-
dard precautions but also to protect nucleic acids from
nuclease degradation (Fig. 15.4). Gloves are absolutely FIGURE 15.5 Gloves are required for working with RNA.
required for handling of RNA (Fig. 15.5). DNA is less
susceptible to degradation from contaminating DNases;
however, repeated handling of samples without gloves RNA on the same bench space. This requires mainte-
will adversely affect the integrity of the DNA over time. nance of RNase-free conditions for all nucleic acid iso-
Standards and controls that are handled repeatedly are lation. In a common isolation arrangement such as this,
the most likely to be affected. Originally, having sep- care must be taken that no inappropriate enzyme expo-
arate areas for DNA and RNA isolation was recom- sures occur if DNA samples are treated with RNase or if
mended. Some laboratories, however, isolate DNA and RNA samples are treated with DNase.
in the refrigerator. Purified DNA can be stored at freezer

Holding and Storage Requirements
temperatures (–20°C to –70°C) in tightly sealed tubes
Methods such as interphase and metaphase fluorescent for up to 10 years or longer. Freezer temperatures are
in situ hybridization (FISH) and karyotyping require preferred for long-term storage; however, a clean DNA
intact cellular structures or culture of cells so that preparation in frequent use is better stored in the refrig-
only fresh specimens are acceptable. Many molecu- erator so as to avoid DNA damage caused by multiple
lar methods, however, do not require the integrity of cycles of freezing/thawing. Shearing of DNA by freeze/
the cellular structure of tissue, only that the nucleic thawing cycles can also occur in a frost-free freezer. As
acid remains intact. In either case, circumstances may previously stated, PCR and methods that do not require
arise that require holding of specimens before analysis. large intact fragments of DNA are more forgiving with
DNA and RNA are stable when samples are collected regard to the condition of the DNA.
and held under the proper conditions. For example, Storage of isolated RNA at room temperature or
multiple tests may be performed on snap-frozen tissue refrigerator temperature is not recommended in the
specimens held at –70°C. Although most amplification absence of stabilization. RNA suspended in ethanol
methods are capable of successful analysis of limit- can be stored at –20°C for several months. Long-term
ing and challenging specimens, Southern or northern storage is best in ethanol at –70°C, although RNA sus-
blot methods will not work consistently on improp- pended in diethylpyrocarbonate-treated water is stable
erly handled specimens or nucleic acids stored under for at least 1 month. As with DNA, the long-term sur-
less-than-optimal conditions. The College of American vival of the RNA depends on the quality of the initial
Pathologists (CAP), which provides accreditation stan- isolation and handling of the specimen.
dards that are followed by CAP-accredited laboratories
to improve the quality of their testing, has published
recommendations for sample and isolated nucleic acid TEST PERFORMANCE
storage (Table 15.2).
For long-term storage, isolated nucleic acid is pre- Various commercial reagent sets or “kits” have become
ferred. Conditions for the storage of isolated nucleic available for patient testing. Many of these reagent sets
acid have been recommended (Table 15.3). Cryotubes are approved by the Food and Drug Administration
and specially designed labels are available for long-term (FDA-approved) for use in clinical testing. The FDA-
nucleic acid storage at ultralow temperatures (Fig. 15.6). approved tests are considered “waived” tests by the CAP
The general rules for holding specimens for proc- and other agencies charged with inspection of medical
essing differ depending on the analyte and its stability in laboratories. For these tests, most of the calibration, val-
the cell. Written procedures indicate the proper handling idation, and other quality assurance measures have been
of specimens for optimal performance of that procedure. established by the manufacturer and approved by the
For example, room-temperature storage is recommended FDA. If the test is performed strictly according to the
for viral RNA in whole blood. Refrigeration of blood manufacturer ’s protocol, implementation of the test only
will cause neutrophils to degranulate, releasing enzymes requires verification of the accuracy, precision, report-
that affect free virus particles. On the other hand, spec- able range, and reference range. If the laboratory mod-
imens in EDTA tubes held at 4oC may not show sig- ifies the protocol, for instance, doing an amplification
nificant loss of human RNA stability, depending on step in a thermal cycler model different from that used
the gene. Still, blood or bone marrow for human RNA to validate the test or performing the test on blood col-
testing should be processed within 24 hours. Blood and lected in a different anticoagulant, then the laboratory
bone marrow specimens sent to outside laboratories for must establish clinical performance.
molecular analysis can be shipped overnight at room Tests in the molecular laboratory that are individu-
temperature or with ice packs. Tissue is best shipped ally designed or are adaptations based on published
frozen on dry ice. methods are called laboratory-developed tests (LDTs)
Isolated DNA of sufficient purity can be stored at and, like modified FDA-approved tests, are consid-
room temperature for several months or at least 1 year ered non-waived for the purposes of laboratory review.
TABLE 15.2 Specimen Storage Requirements for Nucleic Acid Extraction‖
Nucleic Acid Sample Temperature Time
DNA Whole blood, buffy coat, 22°–25°C 24 hours

bone marrow, fluids 2°–8°C 72 hours*
–20°C At least 1 year*
–70°C More than 1 year*
Tissue 22°–25°C Not recommended
2°–8°C Up to 24 hours†
–20°C At least 2 weeks
–70°C At least 2 years
Microorganisms in culture 22°–25°C 24 hours‡
2°–8°C 72 hours‡
–20°C 2–4 weeks‡
–70°C More than 1 year
Cell lysates in GITC 22°–25°C 1–2 weeks§
RNA Whole blood, buffy coat, 22°–25°C Not recommended§

bone marrow, fluids 2°–8°C 2–4 hours*
–20°C 2–4 weeks*
–70°C More than 1 year*
Fluids collected in specialty 22°–25°C 5 days
RNA protection tubes 2°–8°C 7 days
–20°C 2–4 weeks
–70°C At least 7 months
Tissue 22°–25°C Not recommended
2°–8°C Not recommended
–20°C Not recommended
–70°C At least 2 years
–140°C (nitrogen vapor) At least 2 years
Cell lysates in GITC¶ 22°–25°C 1–2 weeks§
Cell lysates in RNA storage 22°–25°C 1 week
solution (Ambion) 2°–8°C 1 month
Microorganisms in culture 22°–25°C 24 hours‡
2°–8°C 72 hours‡
–20°C 2–4 weeks‡
*Separation of white blood cells is recommended to avoid hemoglobin released upon hemolysis of red blood cells.
†
Tissue types differ in stability and nuclease content.
‡
Nucleic acid from cultured organisms is best isolated immediately on harvesting fresh cultures.
§
RNA status depends on the type of cell or tissue and the gene under study.
‖
Depending on gene expression, adequate RNA may be isolated within a few hours. Storage of cell lysates in a stabilizing buffer is best for maintaining RNA.
¶
GITC = guanidine isothiocyanate.
TABLE 15.3 Nucleic Acid Storage Requirements
Nucleic Acid Matrix Temperature Time
DNA TE* buffer or DNase-free water 22°–25°C Up to 4 months
Freeze-dried or dried on collection paper 22°–25°C More than 15 years
TE buffer or DNase-free water 2°–8°C 1–3 years†
TE buffer or DNase-free water –20°C At least 7 years
TE buffer or DNase-free water –70°C More than 7 years
RNA TE buffer or RNase-free (DEPC‡-treated) water 22°–25°C Not recommended
TE buffer or RNase-free (DEPC-treated) water 2°–8°C Not recommended
TE buffer or RNase-free (DEPC-treated) water –20°C Up to 1 month

§
RNA storage solution (Ambion) –20°C More than 1 month
Ethanol –20°C More than 6 months
TE buffer or RNase-free (DEPC-treated) water –70°C Up to 30 days
Ethanol –70°C More than 6 months
*10 mM Tris, 1 mM EDTA, pH 8.0.

†
One year for Southern blot.
‡
DEPC = diethyl pyrocarbonate.
§
1 mM sodium citrate, pH 6.4.
Development of new tests in the clinical laboratory

requires validation of the performance of the method
and reagents in accurately detecting or measuring the
analyte.9
Clinical test parameters must be established from
clinical trial data or published population studies. The
positive predictive value and negative predictive value
of the analyte status in a given disease state justify the
use of the test for patients. The positive predictive value
is the degree that a particular test result corresponds with
the presence of a clinical state, such as disease symp-
toms or response to a therapeutic agent. The negative
predictive value is the clinical specificity of the test, that
FIGURE 15.6 Cryotubes with tight-fitting lids are recom-
mended for long-term freezer storage of DNA and RNA.
is, the absence of that test result in the absence of the
disease state or response.
Analytical test performance is assessed by several
criteria (Table 15.4). Analytical test accuracy is a func-
tion of the sensitivity and specificity of the test.
TABLE 15.4 Measurements of Test Performance
Criteria Definition Example
Analytic sensitivity Change in assay response with All positive reference standards tested positive with the
corresponding change in analyte new assay. The analytical sensitivity of the assay is 100%.
Clinical sensitivity Ability of test result to predict a clinical Of 100 patients with a gene mutation, 95 have a disease
condition state, a clinical sensitivity of 95%.
Detection limit, limit Least detectable presence of the analyte The t(14;18) translocation test can detect 1 translocated cell
of detection in 10,000 normal cells, a detection limit of 0.01%.
Analytic specificity Ability to detect only the analyte and not The Invader assay for factor V Leiden successfully detected
nonspecific targets mutations in 18 positive specimens while yielding negative
results for 30 normal specimens (no false positives).
Clinical specificity Disease-associated results only in patients Of 100 normal specimens, 1 displayed a gene mutation
who actually have the disease conditions (1 false positive), a clinical specificity of 99%.
Precision Agreement between independent test A quantitative method yields 99 results less than 1 standard
results deviation apart in 100 runs, a precision of 99%.
Reproducibility Consistency of test results produced from A qualitative method yields 100 positive results when
the same procedure performed in 10 independent laboratories, a reproducibility
of 100%.
Analyte The range within which a specimen may A qPCR HSV assay yields reproducible linear results from
measurement range be measured directly (without dilution or 10 to 107 copies of HSV per 20 μL of CSF. Specimens within
concentration) this range are measured directly.
Reportable range The range within which test results are A qPCR HCV assay yields reproducible results from
considered to be valid (with or without 15–100,000,000 IU/mL of blood, requiring dilution for levels
dilution) greater than 100,000 IU. Specimens within this range are
reportable.
Reference range Expected analyte frequency or levels from The reference range for prostatic specific antigen is
a population of individuals 0–4 ng/mL.
Analytic accuracy Production of correct results Of 100 specimens with mutations in the HCM gene, 99 are
detected by sequencing, with no mutations detected in
normal specimens.
Linearity Quantitative correlation between test A graph of test procedure results versus input analyte yields
result and actual amount of analyte a straight line.
The analytical sensitivity of an assay equals The analytical accuracy of an assay equals
TP TN + TP
TP + FN ×100 TN + TP + FN + FP ×100
The analytical specificity of an assay equals where TN = true negative, TP = true positive, FN = false
negative, and FP = false positive.
TN True positive and true negative are determined using
TN + FP ×100 a gold standard, such as an established reference assay
or a definitive clinical diagnosis. Note that the clinical resist variations in testing conditions, such as reagent
sensitivity is different from the analytical sensitivity, lots, test days, different technologists, or other sources
which may also be reported (Table 15.4). The analyt- of random error. A very precise test on one instrument
ical sensitivity is defined as the ability of the assay to in one lab on a particular day may not be reproducible
detect the presence of the analyte, with changes in the when performed on a different instrument or with a dif-
assay response corresponding to changes in the amount ferent test lot.
of analyte present. Analytical sensitivity can be further The purpose of test validation is to demonstrate that a
refined by determining the detection limit or lower limit procedure is ready to be implemented as a clinical test.12
of detection. The detection limit is the lowest level of Test validation is performed on specimens of the types
analyte that is consistently detected by the assay. In that will be encountered in the routine use of the test,
qualitative assays, the analytical sensitivity and the limit such as frozen tissue, paraffin-embedded tissue, body
of detection are practically equivalent. In quantitative fluids, and cultured cells. The number of specimens
assays, the analytical sensitivity is defined by differ- tested varies with the procedure and the availability of
ences in quantitative response with quantitative differ- test material. Archived specimens are often used for this
ences in the reference analyte. For tests with quantitative purpose. The results from the new test are compared with
results reported qualitatively (e.g., positive/negative) a those of established procedures that may have been per-
cut-point quantity must be established, above which is formed on these specimens or with the clinical diagno-
considered positive and below which is considered neg- sis. A standard form may be designed for the preparation
ative. Thus, the quantity may be determined empirically of reaction mixes by the test parameters determined in
or statistically using probit analysis.10,11 These criteria, the validation process. Laboratory information systems
along with the analytical measurement range (AMR; are also programmed to produce setup forms for valida-
Table 15.4), are documented as part of the test validation and routine patient testing.
tion process. Predeveloped and FDA-approved molecular methods
The AMR is established by showing accurate test are increasingly available. According to the FDA, an
results on a range of values or test results. The vali- approved test is one that has been accepted by the FDA
dation should include sufficient validation samples at as safe and effective for its intended use, based on the
AMR levels in order to demonstrate performance. For manufacturer ’s data. The manufacturer conducts studies
qualitative results, such as positive/negative or homozy- to show that the test performs as claimed and does not
gous wild-type, homozygous variant, or heterozygous, present an unreasonable risk. The test is offered for sale
sufficient numbers of each type of sample must be avail- after a premarket-approval application has been reviewed
able. For array analyses or next-generation sequencing, and approved by the FDA. A test that has been cleared
all possible alleles do not have to be verified; however, by the FDA has been shown to be similar to other tests
the ability to accurately detect an adequate sampling of already marketed, based on the manufacturer ’s data. For
alleles in a range of concentrations (for somatic variants) such a test, the manufacturer submits comparison results
should be established. Actions taken for samples above in a “premarket notification” that the FDA reviews and
or below the AMR should be documented in the vali- determines that the tests are substantially equivalent.
dation record. Such results may be reported as “greater When FDA-approved or FDA-cleared methods are
than” or “less than” the limits of the AMR, or the vali- incorporated, the test performance is verified by using
dation may include methods and criteria for dilution or the purchased reagent sets to test validation specimens.
concentration of samples. Note that dilution and concen- This verification establishes that the results of the com-
tration do not increase the AMR. mercial test performed in the individual laboratory are
Other criteria for validation include precision and as predicted by the developer. If the commercial test is
reproducibility. Precision is the correct qualitative result modified, validation is required to show equal or supe-
or the degree of variation in a test of quantitative results rior performance of the modified procedure.
when performed on the same sample multiple times. Once a procedure has been established, the method
Precision should be established throughout the report- is documented in the laboratory according to CLSI
able range. Reproducibility is the ability of the test to guidelines. The procedure description should include
detailed information, for instance, primer and probe regions where variant calls can be affected by sequence
sequences, their purification conditions, and labeling. structure, such as highly homologous regions or long
A copy of the standard form used to set up reaction nucleotide repeats, the laboratory must document how
mixes is included in the procedure description. A clear interference is mitigated or avoided.
description of formulas and reporting units is required Performance characteristics, including sensitivity,
for quantitative results. Interpretation of qualitative data, specificity, and precision (reproducibility), for variants
acceptable ranges (e.g., band patterns), product sizes, cover examples of the different variant types (single-
melting temperatures, and reasons for rejecting results nucleotide variants, indels, copy-number and other struc-
are required information. Methods used to score FISH or tural variants) to be detected. The limits of detection for
array results relative to internal control loci are also part variants in samples are established for heterogeneous
of the written procedure. It is useful to incorporate pic- genotypes (e.g., heterogeneous tumor samples or micro-
tures of gel patterns or instrument output data showing chimerism). For somatic analyses of solid tumors, the
positive, negative, heterozygous, or other reportable sensitivity and limit of detection will be affected by the
results. tumor cell percentages in the tested tissue. This informa-
The clinical accuracy of a test is determined by cor- tion is also documented.
relation of the test results with morphological, radio- The detection rate of variants by bioinformatics proc-
graphic, or other clinical data. For rare conditions or esses and software is applicable to exome and genome
where clinical associations are well established, liter- sequencing data analysis for suspected genetic disease
ature reports of correlation with the analyte state may components. Acceptance and rejection criteria for the
be used. The indications for ordering the test are estab- results generated by the analytical bioinformatics process
lished based on the clinical utility as determined by the are based on quality control (QC) parameters established
validation process and are documented in the procedure during instrument and software optimization, including
manual. For forensic testing, all aspects of the test, from base and mapping quality scores, read coverage, and
validation to test reporting, should adhere to guidelines numbers and types of variants.
established by the DNA Advisory Board Standards The type and quality of sequence databases used for
and the Scientific Working Group on DNA Analysis annotation (identification of the significance of variants)
Methods. are established as part of the validation process. Data-
The procedure manual or standard operating proce- bases may be constitutional for genetic diseases, popu-
dure is maintained in the laboratory and reviewed at lation based to identify the frequencies of minor alleles,
least annually. If a test is discontinued, the written pro- or cancer specific. Clinical laboratories may establish
cedure, noted with the dates of initial use and retirement, internal databases as well.
is kept for at least 2 years. Some laboratory profession-
als maintain retired procedures for longer periods.
Calibrators and Method Calibration
Calibration is the establishment of conditions for
Next-Generation Sequencing
instrument response/method result association with
Validation parameters for NGS will vary between tests the true value of quantitative analyte measurements.
and typically include a description of the analytical These values are represented in calibrators. Calibrators
target (exons, genes, or targeted regions, such as introns are samples of known amounts/concentrations of mol-
or promoter sites) and bioinformatics used for analysis. ecules of the same type measured in the assay, such
Sample pooling methods are designed such that indi- as an admixture of PCR products in known proportion
vidual sample identity is maintained throughout testing or an RNA transcript calibrator in a matrix of normal
and analysis. Criteria and thresholds for variant calling RNA. Calibrators can be purchased from manufactur-
include minimum read coverage depth, base or variant ers. Some are supplied with instruments for instrument
quality scores, and allelic read percentage. These criteria maintenance. Others are supplied with reagent sets for
may be different based on application (e.g., detection of validation or re-validation purposes. Patient specimens
germline versus somatic mutations). If there are target previously tested by a validated method or specimens
used in proficiency testing are also calibrators. When a be established by repeated runs of each control sample
calibrator is in a different matrix (diluents or mixture) on different days by different personnel. The average
than the samples to be tested, such as with general- of ±2 or ±3 standard deviations defines the acceptable
purpose commercial calibration sets, commutability range for positive controls. A set of controls may also
(lack of matrix effects) must be established.13 be included with runs in the form of a calibration curve
Calibrators covering the AMR demonstrate linearity with each run.
if calibrator values are plotted against instrument or test Real-time PCR methods that automatically determine
output values. In the test validation, the AMR is estab- analyte levels require measurement of a standard curve
lished with samples of the appropriate type and matrix. or dilution series of analyte levels encompassing the
Calibrators may not necessarily be supplied in the same levels expected from the patient specimens. On some
matrix but can be diluted or dissolved into the appro- instruments, the standard curve must be run simultane-
priate matrix. The source, quality, and preparation of ously with the specimens, whereas in others, previously
the calibrators should be documented. Certificates of determined curves may be loaded into the software.
quality from the vendor accompany commercially sup- Alternatively, results can be calculated manually by
plied calibrators. Re-calibration is performed if calibra- linear regression of the test results, using standard-curve
tion does not meet the required standards of linearity data in spreadsheet software.
or accuracy. In methods requiring detection of a target-specific
Calibration verification should be done at regular product or relative amounts of target, internal con-
intervals, at least every 6 months, or at intervals estab- trols are run in the same reaction mix as the test spec-
lished by the laboratory. Calibration verification may imen. For example, RNA from housekeeping genes is
be required upon changes of or major repairs to instru- used for internal controls in methods quantifying infec-
ments or changes in reagent lots that might affect test tious agents or detecting tumor cells by expression of
performance or when quality control indicates shifts or tumor-specific transcripts.14 Centromere-specific probes
unacceptable errors in test control results. Verification serve as internal controls in FISH analyses, as do house-
should demonstrate the continued linearity of correlation keeping gene probes on microarrays. The presence of
between the calibrator values and test results. an internal control supplies a base for normalization
of results. In PCR, the internal control distinguishes
false-negative results from failed amplifications. Internal
Controls
controls that are amplified in the same tube with sample
Controls are samples of known type or amount that templates are designed to not interfere with or inhibit
are treated like and run with patient specimens. Inter- target amplification, which could yield a false-negative
pretation of test results always includes inspection of result. Failed internal controls are documented and call
controls and standards to verify acceptable test perfor- for a repeat of the assay.
mance. With qualitative tests, a positive, a negative, and, The controls and standard curve should cover the
in some cases, a sensitivity control are required. The critical detection levels or results of the method. Control
sensitivity control defines the lower limit of detection results are continually monitored to spot trends or spikes
for more meaningful interpretation of negative results. outside of tolerance limits. Coefficients of variance or
For nucleic acid amplification techniques, an ampli- standard deviations of quantitative control levels should
fication control is used. The amplification control is also be calculated at regular intervals. Laboratory pro-
a target that should always amplify. The amplification fessionals may establish criteria for control tolerance
control distinguishes true-negative amplification results limits and document actions to be taken in the event of
from false negatives resulting from amplification failure. an unacceptable control result.
In quantitative methods, high-positive, low-positive, and Control probes for arrays and in situ hybridization
negative controls are included with each run. The high methods depend on the signal pattern to be tested (ampli-
and low levels should be similar to critical points in the fication, structural change, or deletion) and the tissue
assay, such as the lowest detectable level of analyte. An being tested. Internal control probes may map to the
acceptable range for high- and low-positive controls may centromere or other locus close to the test probe–binding
site. External controls, probes run on specimens in par- QUALITY ASSURANCE

allel to the test specimen, are used for loci that may not
have an internal control, such as the Y chromosome Monitored controls and conditions are reviewed monthly
in females. Controls for most assays are commercially or at determined intervals to document outliers or sys-
available or supplied with reagent sets. When commer- temic error. These monitors can be established by the
cially prepared controls are not available, alternative IQCP. Periodic review and documentation of test results
controls (previously tested samples tested in duplicate, are required for all clinical testing, including molecular
samples tested by a different method, or samples tested tests. Review might be, for example, in the form of rates
in independent laboratories) are used. Nucleic acid from of positive and negative results compared with expected
these controls is best prepared in larger quantities, ali- rates from independent sources, such as published
quoted, and stored in conditions where they are most results, over time. This type of monitoring reveals trends
stable. Just as with new lots of other reagents, new ali- or shifts in the rates of positive or negative results. Crit-
quots are tested with old aliquots to verify consistent ical values that require physician notification are estab-
control results, and the procedure for the use and storage lished by validation and confirmed by monitoring.
of all controls is documented. As with other types of quantitative testing, defined
dynamic ranges, sensitivity levels, and accuracies are
subject to review. For instance, a test that determines
QUALITY CONTROL viral types by melt curves must include a defined
narrow temperature range for the Tm of each viral type
Positive, sensitivity, and negative controls should be (Fig. 15.7). These values are established during the test
included in each run of patient samples. For tests with validation and should be reviewed periodically using
multiple targets, controls can be systematically rotated high, low, sensitivity, and negative standards in each
for different targets in each run. Controls for quantita- run. Band patterns, melt curves, and peak characteristics
tive tests should reflect the clinically critical levels of should be defined with regard to how the results of the
test results. Alternatively, the laboratory can establish test are to be interpreted.
an individualized quality control plan (IQCP). This plan Assay levels that distinguish positive from nega-
includes monitoring of the extraction and amplification tive results (cut-off values or cut points) must be well
steps of the assay. Tests with electronic or built-in con- defined and verified at regular intervals. In assays such
trols are appropriate for the IQCP. as single-strand conformation polymorphism, in which
Tolerance or acceptability levels of control values control patterns are not identical from run to run, normal
should be established based on the validated precision
and reproducibility of the assay. Monitoring can be
performed using Levey–Jennings or modified Levey–
Jennings plots15 or cusum plots,16 and tolerance limits
can be expressed as Westgard rules.17 The results of Strain A Strain B
these monitors are reviewed before reporting test results.
Review takes place at least monthly and may include
multiple systems and networked laboratories.18 If the dF/dt
control results violate the set limits, then corrective
action (repeating the run with replacement controls) has
to be taken and documented. If patient specimen avail-
ability precludes repeating a run, further investigation 40 80
into the nature of the control failure and its effect on the Temperature (°C)
patient result may allow acceptance of the sample results FIGURE 15.7 Tm ranges defined for two strains (A and B) of
without repeating. Comparison of test values with previ- a theoretical microorganism are nonoverlapping. The melt
ous results or historical averages may serve to increase curve shown would indicate that the test specimen contains
confidence in the test results. strain A.
controls for each scanned region are included in every INSTRUMENT MAINTENANCE
run. Quantitative results should be within the linear
range of the assay. The linear range is established by Instruments used in the molecular laboratory must be
measuring dilutions or known concentrations of standard monitored and maintained for consistent performance
and establishing a direct correlation (standard curve) and accurate test results. Manufacturers supply recom-
between test output and standard input. The technolo- mendations for routine maintenance. Service contracts
gist may observe that raw data are consistent with the are used to provide support from trained service tech-
final interpretation of the results. For example, if a viral nicians. Laboratory professionals maintain a schedule
load is interpreted as negative, the raw data should be and instructions for all routine maintenance, such as
below the cut-off value established for the test. Calcula- checking temperature settings, timers, and signal back-
tions and comparisons with standards used to verify test ground levels. Parts are replaced as required or specified
results should be described in the laboratory procedure by the instrument manufacturer. Maintenance schedules
manual. should reflect the amount of use of the instrument. Most
For sequencing procedures, information about the routine maintenance and minor troubleshooting, such
genes tested and variants found is continually updated, as replacing bulbs or batteries, may be performed by
along with the databases used for annotation. Docu- the technologist with the aid of clear instructions from
mentation of the method and results of verification of the manufacturer or as prepared by laboratory manage-
genetic or germline variants found is maintained by the ment. These instructions must be readily available to
laboratory. the technologist in the event of instrument malfunction.
When controls or results exceed acceptable limits, Technologists should be aware of the limits of user-
corrective action is taken. Corrective action can be recommended repairs and when service calls are indi-
minor adjustments or a repeat of a sample or run or more cated (Fig. 15.8). Laboratory professionals document
extensive changes to a system. Documentation of what all maintenance, service calls, calibrations, and part
is done in corrective action reports is an important com- replacements.
ponent of quality assurance. Refrigerators and freezers used to store patient mate-
Quality assurance also includes documentation and rial and reagents are monitored at least daily (Fig. 15.9).
maintenance of laboratory procedures and methods. Maximum/minimum thermometers register the highest
These procedures are frequently updated with events and lowest temperature reached between monitoring
ranging from major advances in instrument technology
to changes in reagent availability. Electronic document
control systems facilitate editing of documents as well
as communication of the status of policies and proce-
dures with laboratory personnel.
Test results may show discrepancies with other lab-
oratory findings, with clinical observations, or with the
laboratory’s own preliminary results. These discrepan-
cies are documented along with any corrective action
taken, if necessary. Due to the nature of molecular
pathology testing (increased sensitivity, high resolu-
tion, varied methodologies used) discrepancies may be
explained by the technical aspect of the test. In these
cases, the test validation should include data on the clin-
ical significance of the analyte as detected or measured FIGURE 15.8 Routine maintenance, such as capillary re-
by the molecular test, for example, detection of analyte placement and instrument cleaning, is performed by the labo-
below levels of clinical significance. Statistics on these ratory technologist. Dangerous or complex maintenance, such
results may also be collected as part of quality assurance as repair or replacement of a laser source, is performed by the
and control. service representatives.
FIGURE 15.9 Certified chamber ther-

mometers are used for monitoring tem-
peratures in incubators, refrigerators,
ovens, and freezers. Thermometers must
be checked at least annually by compari-
son with a NIST-calibrated thermometer.
“High-low” thermometers or commercial
monitoring systems are used when moni-
toring is required when the laboratory is
not in active operation.
points. During a busy shift, refrigerators and freezers checked periodically for proper temperature control.
may be opened frequently, causing the temperature to Thermometers with flexible probes (type K thermocou-
increase temporarily. This must be taken into account ples) are convenient for checking representative wells in
while monitoring. Out-of-range temperatures (e.g., more a block thermal cycler (Fig. 15.10). Approaches differ
than ±2°C of the set temperature) are recorded. Con- regarding whether each well should be checked with
firmatory temperature checks at different times during each routine measurement or whether representative
the shift are required before further action is taken. wells should be checked, with different wells checked
Heat blocks, incubators, ovens, and water baths are also each time. In some laboratories, test reactions are run
monitored for temperature stability and accuracy. U.S. in different wells to demonstrate successful amplifica-
National Institute of Standards and Technology (NIST)– tion at each position on the block. More thorough and
certified standard thermometers are used for this type of accurate temperature measurements are achieved with
monitoring. Thermometers verified by a NIST thermom- computer systems, with fixed or flexible probes designed
eter are also acceptable for this purpose. Specialized dry to measure the temperature in all wells throughout a
bath thermometers, which are encased in a microfuge PCR program, including ramping of the temperature
tube of mineral oil, or electronic temperature probes are up and down, overshooting set temperatures, and tem-
used to monitor heat blocks. All storage and incubation perature drift during the hold phase of each step. Non-
equipment is kept clean and free of contaminated spec- block thermal cyclers, such as air-heated or modular
imens and expired reagents. If out-of-date reagents are instruments, are tested with probes modified to fit as
used for research or other purposes, they should be well the capillaries or tubes used in these instruments. Real-
marked and/or maintained in a separate area from the time thermal cyclers require additional maintenance of
clinical test reagents. Although standard thermal cyclers the detection system. Manufacturers supply materials to
have no automated moving parts, they may decline in check for “bleed through” of fluorescence of different
temperature control over time. This is especially true of wavelengths. Background measurements are made using
block cyclers where hot and cold spots develop within water or buffer samples. Each laboratory will establish
the sample block. For this reason, thermal cyclers are the type and frequency of scheduled maintenance.
FIGURE 15.10 Block thermal cyclers may be monitored

using a thermometer with a flexible probe. More thorough
monitoring is performed with multiple temperature probes and
software that follows the ramping temperatures as well as the
holding temperatures.
FIGURE 15.12 Gel electrophoresis equipment must be main-

tained free of precipitate and properly handled to avoid shock
exposure.
balance, have been used for many years. The weights

are converted to volumes. The mean of several measure-
ments from the same pipet reveals its accuracy. The stan-
dard deviation or coefficient of variance is calculated to
FIGURE 15.11 Centrifuge speeds are checked at least annu- determine the degree of reproducibility (imprecision) of
ally, and the results of actual and set speeds are posted on the the pipet. Service providers will clean and check pipets
instrument. on a per-pipet charge.
Electrophoresis power supplies are tested at least
Centrifuges and microcentrifuge speed controls are annually to ensure delivery of accurate voltage and
monitored at least annually using a tachometer. In some current. Personnel should be trained in safe operation.
institutions, technologists perform this calibration. Alter- Leads and connectors to gel baths are kept free of pre-
natively, an institutional engineering department may cipitate (Fig. 15.12). Salt precipitation can be avoided
provide this service. The actual speed of rotation is deter- by not leaving buffer in gel baths after electrophoresis
mined and recorded, along with the set speed or setting runs. Capillary systems require cleaning of buffer and
number on the centrifuge. This information is then posted polymer delivery channels as well as replacement of the
on the instrument (Fig. 15.11). Automatic pipettors used polymer at least twice per month or as required per its
for dispensing specific quantities of reagents are checked expiration date. Capillaries are also replaced according
for accuracy before use and at 6-month intervals or as to their suggested life span in number of uses or time
required according to use. Gravimetric methods, where in use. Temperature-controlled electrophoresis equip-
measured samples of water are pipetted to an analytical ment, such as capillary systems and those used for
constant-temperature gel electrophoresis, is monitored acceptable background levels. Laboratory profession-

for accurate temperature settings as recommended by als may use these measurements or establish their own
the manufacturer. acceptable background levels. Corrective action, such as
Photographic equipment is frequently used in molec- cleaning or filter adjustment, is documented in the lab-
ular laboratory procedures. Autoradiograms resulting oratory maintenance records. Ultraviolet (UV) illumina-
from radioactive or chemiluminescent methods are tors are kept free of dust and properly shielded while in
developed in automated equipment or manually. The use. The technologist keeps track of the life span of the
processing equipment is maintained with fresh fixing UV light source and replaces it accordingly.
and developing solutions free of debris or sediment. Signal response from spectrophotometers used to
Digital cameras are rapidly replacing Polaroid cameras measure DNA, RNA, protein concentrations, and colo-
for documenting gel data. Cameras should be firmly rimetric assays and turbidity is checked annually or as
mounted and adjusted for optimal recording of gel data, recommended by the manufacturer. Maintenance also
free of shadows, dust, and other photographic artifacts includes scanning through the range of wavelengths
(Fig. 15.13). used (e.g., 200 to 800 nm) with supplied materials or
Periodically, background measurements are required filters. Operation manuals will include instructions for
on such instruments as fluorometric detectors (including calibration and maintenance.
real-time thermal cyclers), luminometers, and densitom- Fume hoods and laminar flow (biological safety)
eters. Instrument manufacturers provide guidelines for hoods (Fig. 15.14) are monitored annually for proper
airflow. Fume-hood testing requires special equipment
and is likely to be performed by building engineers.
FIGURE 15.13 Cameras should be mounted securely for gel

photography. A digital camera, shown here, is mounted on a FIGURE 15.14 Laminar flow hoods are used to protect
photographic hood. A mask shields the ultraviolet light source against biological hazards and to help maintain a sterile
except for the area where the gel is illuminated. environment.
Laminar flow hoods are tested for proper filter perfor- REAGENTS
mance and air displacement. This testing is performed
at least annually or upon installation or movement of the When reagents are replaced in a test method, the new
hood. Professional service technicians or the hood man- lot is ideally tested on a previously positive and nega-
ufacturers usually provide this type of certification. tive specimen as well as the run controls. Instructions
For all detection systems, regular monitoring of on the preparation of reagents and the quantities used in
functional characteristics will reveal any drift or trends each assay are included in the written laboratory proto-
that might affect test results. Tolerance limits should be col for each procedure. Lot numbers and working stocks
established to warrant intervention by maintenance or of probes and primers used in amplification methods
recalibration of the instrument. Scheduled and unsched- are documented and matched to test performance in the
uled maintenance is documented and kept in the labora- runs in which they were used. The sequences of primers
tory records. These records should be readily available and probes are also documented because any sequence
to the technologists using the equipment. errors made during ordering or synthesis of the primers
will adversely affect the amplification specificity or even
Instrument Calibration result in amplification failure. Probes used for linkage
analysis and array technology are periodically updated
Calibration is fitting an instrument or test system output as new markers are discovered, so the probe sequences
with the actual concentration of a reference analyte by used for a given test should be recorded.
testing and making appropriate adjustments. In cali- Primers are a critical component of PCR procedures.
bration verification, materials of known concentration Primers are most conveniently supplied in lyophilized
throughout the reportable range are tested as patient (freeze-dried) form from the DNA synthesis facility
samples to ensure the test system is accurate. If calibra- (Fig. 15.15). The supplier will also provide information
tion verification fails, recalibration is required. CLIA-88 on the quality, method of purification, molecular weight,
regulations, 42 CFR 493.1255(b)(3), recommend the
performance of calibration verification at least every
6 months or when major components, instrument soft-
ware, or lots of reagents of the test system are altered.
Recalibration is also required if proficiency or other
quality control testing fails or in the event of major
instrument malfunction and repair. Manufacturers of
test systems may also provide calibration schedules and
instructions on how to perform calibrations. Laboratory
professionals must verify the calibration of systems per-
formed by the manufacturer.
A variety of materials may be used for calibration,
including previously tested specimens, reference stan-
dards, commercial calibrators, and proficiency testing
material. There must be an independent assessment of
the actual measurement of the calibration material. Once
established, calibrator results should always be specified
ranges of values. Calibration materials should cover at
least three levels of measurement: low, medium, and
high points. Analytes used for calibration should be in FIGURE 15.15 Primers are purchased from DNA synthesis
the same matrix (e.g., plasma or urine) as the patient facilities. On receipt in freeze-dried form, the primers are
specimens.19 Calibrators are prepared and used sepa- easily resuspended in nuclease-free water or buffer to make a
rately from quality control standards (e.g., positive, neg- stock solution. The stock solution is then diluted into working
ative, sensitivity controls) for routine runs. stocks.
and number of micrograms of dried primers. This infor- reports. Labeling methods and standards for adequacy of
mation is used to rehydrate the primers to a stock solu- hybridization are included in the test procedure manual.
tion concentration required for the PCR protocol. The In multiplex reactions, primer and/or probe competi-
resuspended primers are then diluted into working tion for substrates may affect the results. Multiple fluo-
stocks. rochrome signals in fluorescence assays may also cross
Probes used for real-time PCR are supplied in solu- into each other ’s detection ranges. For gel or capillary
tion or in dried form, for example, a 100-μM supplied or sizing, products of multiplex reactions should be reason-
resuspended stock solution that is diluted to 4- or 5-μM ably different so that banding patterns do not complicate
working stock before use in the procedure. When new interpretation. For example, multiplex STR analysis by
working stocks are prepared (diluted from the probe PCR includes 13 sets of primers that produce 13 ampl-
stocks or resuspended primers), they are treated as new icons labeled with three different fluorochromes. The
reagent lots. Master mixes of primers, probe, buffer, range of sizes of each amplified STR locus is designed
nucleotides, and enzyme may be prepared or purchased not to overlap others labeled with the same fluoro-
and used as working stock. chrome. The instrument that detects the fluorescence is
As is required for all reagents, instructions on prepa- also calibrated to subtract any overlap of detection of
ration of primers, probes, and working stocks, along one fluorochrome with another.
with the sequences and binding sites of primers and
the expected size of the amplicons, are documented
Reagent Categories
as part of the written laboratory protocol. Polymor-
phisms or translocation breakpoints that affect primer The Medical Device Amendment to the Federal Food,
binding should be noted in terms of the expected fre- Drug and Cosmetic Act (1938) authorized the FDA to
quency in the population or in the number of successful regulate medical devices, including laboratory tests.
amplifications. Subsequent acts and amendments have further defined
For hybridization procedures, labeled probe solu- the control, risk, safety, and performance of medical
tions are treated as working stock and verified by par- devices. A growing list of categories defines the degree
allel analysis with old lots. FISH probes are validated of endorsement of the performance of tests and test com-
and verified according to recommended procedures. The ponents by the FDA.
quality of new microarray lots is verified by the manu- Analyte-specific reagents (ASRs) are probes,
facturer or by hybridizing labeled nucleotides that bind primers, antibodies, and other test components that
to all probes on a representative array from the lot. RNA detect a specific target, such as a cell surface protein
probes are maintained under RNase-free conditions to or DNA mutation. ASRs comprise the active part of
protect their integrity. “homebrew” tests and are usually purchased from an
Descriptive information on probes is documented outside manufacturer. ASRs are classified as I, II, or III.
in the laboratory. This information includes the type Most ASRs used in the molecular laboratory are class I.
of probe (genomic, cDNA, oligonucleotide, plasmid, Several molecular tests are available as ASRs in infec-
or riboprobe) and the species of origin of the probe tious disease, tissue typing, and other areas of molecular
sequence. The sequence of the probe, a GenBank diagnostics. Approved molecular methods include tests
number or other identification of the target sequence or that utilize FISH, hybrid capture, PCR, and microarray
gene region recognized by the probe, and a restriction technologies. Class II and III ASRs include those used
enzyme map of that region are also important informa- by blood banks to screen for infectious diseases and
tion. Any known polymorphisms, sites resistant to endo- those used in the diagnosis of certain contagious dis-
nuclease digestion, and cross-hybridizing bands should eases, such as tuberculosis. Class I ASRs are not subject
be noted. Recombination frequencies and map positions to special controls by the FDA. The test performance
must be documented for linkage procedures. For inher- of class I ASRs is established during the validation of
ited disease tests, the chromosomal location of the target individual LDTs.
gene and known mutant alleles and their frequencies In the diagnostic laboratory, in vitro diagnostic
in various ethnic groups might be cited in published (IVD) devices or reagent sets are intended for use in the
diagnosis of disease or other conditions. IVD reagent sets

include products used to collect, prepare, and examine
specimens collected from patients. They should be used
strictly according to the manufacturer ’s protocol.
IVD devices are classified according to performance
risk and requirements for surveillance and verification,
from class I (lowest risk) to class III (highest risk).
Regardless of risk level, the performance (accuracy, pre-
cision, analytic sensitivity, reportable range, reference
range) of IVD tests must be verified by the laboratory in
which they will be used. If an IVD protocol is modified,
the procedure must be validated to show equivalent or
better performance compared with the original assay. A
list of currently available FDA-approved tests is located
on the Association for Molecular Pathology website
(https://www.amp.org/).
The FDA would approve the analytical validity
only of a proposed category of in vitro analytical test
(IVAT) reagents, with no claims of clinical utility. The FIGURE 15.16 Flammable and explosive materials are
clinical laboratory would be responsible for any clini- stored in designated protective cabinets or explosion-proof
cal claims. This category is intended to accommodate refrigerators.
the use of promising high-complexity technologies
facing long clearance processes; research use only radiation are increasingly being replaced by nonradio-
(RUO) and investigational use only (IUO) reagents active alternatives, some laboratory procedures still
are not intended for diagnostic use. RUO reagents are use these agents. The Nuclear Regulatory Commission
not intended for use on patient samples, whereas IUO requires that laboratory personnel working with radio-
reagent can be used on patient samples with proper insti- active reagents maintain a radiation safety manual pro-
tutional review and informed consent, for example, in viding procedures for the safe handling of radioactive
clinical trials. RUO and IUO reagents are used to gather substances in both routine and emergency situations.
data that may result in the advancement of a product’s The Occupational Safety and Health Administration
status. These types of reagents are used for testing stan- (OSHA) has also developed regulations regarding ioniz-
dard analytes, such as viral load. ing and nonionizing radiation.
Radioactive reagents and methods are performed in
designated areas. Working surfaces are protected with
Chemical Safety
absorbent paper, drip trays, or other protective con-
Volatile and flammable reagents used in the molecu- tainers. Potentially volatile radioactive materials are
lar laboratory, such as xylene, methanol, ethanol, and handled under a fume hood. Radioactive waste is dis-
isopropanol, are stored in properly vented and explo- carded in appropriate containers, separate from normal
sion-proof cabinets or refrigeration units (Fig. 15.16). trash, according to regulations. Some isotopes with short
The National Fire Protection Association (NFPA) has half-lives may be stored over approximately seven half-
developed a series of warning labels for universal use on lives, checked for residual emissions, and then discarded
all chemical containers (Fig. 15.17).20 Secondary or rein- with regular waste. Containers and equipment used in
forced containers are required for transport and handling these areas should be labeled with “Caution Radioactive
of dangerous chemicals, such as concentrated acids and Material” signs (Fig. 15.18). Signs should be posted in
phenol. the rooms where radioactive materials are used. OSHA
Radioactive chemicals are used in some molecu- has specifications for accident prevention signs and tags
lar methods (Table 15.5). Although methods involving for radiation and other occupational hazards.
HAZARDOUS MATERIALS (see Table 15.5), so exposure at close distance, such as

CLASSIFICATION working over open containers, should be avoided. For
isotopes such as 32P, acrylic shielding is required for
HEALTH HAZARD FIRE HAZARD
Flash Point work, storage, and waste areas (Fig. 15.19).
4 Deadly 4 Below 73°F
3 Extreme Danger 3 Below 100°F
2 Hazardous 2 Below 200°F
Reagent Storage
1 Slightly Hazardous 1 Above 200°F
0 Normal Material Proper reagent storage ensures optimal stability and
2
0 Will not burn
assay performance. The time and temperature of storage
should be documented. Reagents for molecular testing
are supplied with recommended storage conditions and
expiration dates. Expiration dates can be recorded in lab-
3 1 oratory documents or on the reagent containers.

Storage conditions should not be altered. Most
enzymes, antibodies, and other proteins are stable at
freezer temperatures (–18°C to –25°C); however, mul-
SPECIFIC
HAZARD
W REACTIVITY
4 May deteriorate
tiple freeze–thaw cycles will degrade the proteins. Poly-
merases, endonucleases, and other enzymes supplied in
glycerol are protected from the formation of ice crystals
Oxidizer OXY
3 Shock and heat that will degrade the proteins. Reagent supplied frozen
may deteriorate
Acid ACID 2 Violent chemical in larger volumes (1 to 50 mL) can be aliquoted to avoid
Alkali ALK change freezing and thawing the entire amount multiple times.
Corrosive COR
Use No Water W
1 Unstable if Because thawing is sometimes done on ice, and even
heated
Radiation 0 Stable
at room temperature, the time required for thawing can
slow the testing process. Reagents to be stored at refrig-
erator temperatures (4oC to 10oC) may not be stable at
freezer temperatures because they may not survive even
FIGURE 15.17 NFPA hazard labels have three parts, labeled a few freeze–thaw cycles. Not all proteins are stable at
with numbers 0 to 4, depending on the amount of hazard, from
freezer temperatures. Frost-free freezers are not recom-
none (0) to severe (4). The fourth section has two categories.
OXY indicates a strong oxidizer, which greatly increases the
mended for reagent and specimen storage because the
rate of combustion. The W symbol indicates dangerous reac- defrosting cycle can thaw small reagent volumes.
tivity with water, which would prohibit the use of water to Freeze-dried or anhydrous reagents are stored at room
extinguish a fire in the presence of this chemical. See Color temperature, unless otherwise specified. In some areas,
Plate 12. humidity may have to be monitored where dry and dried
reagents are stored.
Laboratory personnel working with radioactive mate-
rial should receive special training for the safe handling,
Reagent Labeling
decontamination, and disposal of radiation. Laboratory
instructions for working with radiation should include In addition to the time and temperature of storage, reagent
inspection and monitoring of shipments as required by labels provide chemical and safety information, includ-
the U.S. Department of Transportation. Workspaces ing the chemical name, signal words (danger or warning),
are decontaminated daily and checked at least monthly a pictogram of hazard symbols, the manufacturer/
by swipe testing or by Geiger counter. Technologists supplier ’s contact information, and precautionary and
wear gloves, lab coat, and safety glasses when han- hazard statements. The United States adopted the Glob-
dling radioactive solutions. Radiation badges are worn ally Harmonized System (GHS) of Classification and
when handling 1.0 mCi or more. Exposure increases Labeling of Chemicals in 2012 as an update to the Occu-
with decreasing distance from the radioactive reagent pational Health and Safety Act. The system was fully
TABLE 15.5 Examples of Radionuclides Used in Laboratory Methods
Radioisotope Half-Life* Radiation (MeV)† Travel in Air Critical Organs

32
P 14.29 days β, 1.709 20 feet Bone, whole body
33
P 25.3 days β, 0.249 20 feet Bone, whole body
3
H 12.35 years β, 0.019 0.65 inches Body water
14
C 5,730 years β, 0.156 10 inches Whole body, fat
35
S 87.39 days β, 0.167 11 inches Whole body, testes
125
I 60.14 days X, γ, 0.035 3 feet Thyroid
*Time for half of the radiation emission to dissipate.

†
Million electron volts.
RADIATION
FIGURE 15.18 Rooms, cabinets, and equipment containing
radioactive chemicals are identified with radiation safety
labels. See Color Plate 13.
implemented in June 2016. This system requires updat-

ing of chemical labeling and standardization of Material
Safety Data Sheets (MSDSs), now designated Safety
Data Sheets (SDSs). SDSs are available to all laboratory
personnel in hard-copy or electronic form. FIGURE 15.19 Acrylic shielding is required for working
Reagents aliquoted or diluted into secondary contain- with gamma emitters, such as 32P and 33P.
ers must also be labeled with the reagent name, hazard
symbols, and, if diluted, concentration and diluent, if
other than water. Primary and secondary labels should
indicate expiration dates.
A chemical hygiene plan for the laboratory should clear bands or peaks without high background, cross-
include responsibilities for lab personnel, chemical hybridization, distortions, and other artifacts. Con-
handling instructions, PPE usage, exposure monitoring trols should also be clear and consistent and reflect the
requirements, and employee training. Signage posted expected size or level. Molecular-weight ladders on gels,
at the laboratory entrance should indicate the presence autoradiograms, or electropherograms should cover the
of carcinogens such as ethidium bromide or radioactive expected range of band or peak sizes produced from
materials, irritants such as xylene, flammable substances the specimen. For example, if primers used to detect
such as reagent alcohol, and other hazards.21 a t(14;18) translocation test by PCR yield amplicons
expected to range from 150 to 500 bp, the molecular-
weight ladder used must range from less than 150 bp to
PROFICIENCY TESTING more than 500 bp.
A record of the assay conditions and reagent lot
Proficiency testing refers to the analysis of external
numbers is kept with patient results. Identification of the
specimens from a reference source supplied to indepen-
technologist performing the assay may also be included.
dent laboratories. Proficiency testing is performed to
Documentation of the quality and quantity of the iso-
assess the skills (competency) of laboratory personnel
lated DNA or RNA is also required, especially if des-
performing molecular assays as well as the performance
ignated amounts of nucleic acids are used for an assay.
of the assay itself. The availability of comprehensive
Quantity and quality are documented in the form of
test specimens in the rapidly expanding area of molec-
spectrophotometry or fluorometry data or quantity or
ular diagnostics is sometimes problematic. The CAP
gel photographs of high-molecular-weight DNA or ribo-
supplies specimens for molecular oncology, engraft-
somal RNA quality. The quality of RNA analyzed by
ment, and microsatellite instability testing, among others
northern blot or RT-PCR may be assessed by monitor-
(http://www.cap.org). Several analytes, however, are not
ing a housekeeping gene, ribosomal RNA expression, or
available, especially for tests that are offered in a small
other calibrator. New lots (or new shipments from the
number of laboratories. If proficiency specimens are
same lot) should be tested with controls before use.
not commercially available, laboratories can exchange
If DNA is cut with restriction enzymes for Southern
blinded split specimens; alternatively, blinded specimens
blot or PCR-restriction fragment length polymorphism,
measured or documented by independent means, such as
complete cutting by the restriction enzyme is verified on
chart review, can be tested within the laboratory. Increas-
control targets and documented by photography of the
ing numbers of reference standards that can be used for
cut DNA on the gel after electrophoresis. DNA digested
proficiency testing are commercially available.22,23 If at
with DNase for array analysis is also documented in
all possible, inter-laboratory testing is preferable.
this way to confirm proper fragment sizes. If specimen
Proficiency testing is performed at least twice a year,
nucleic acid is labeled for hybridization arrays, label-
with the proficiency samples tested within routine patient
ing efficiency is assessed by measurement of the spe-
runs. Handling, analysis, review, and reporting of profi-
cific activity (signal per ng nucleic acid). For Southern
ciency test results are included in a written laboratory
blots, patient identification, gel lane (well) number, and
proficiency testing procedure. The specific procedures
probe target and type are also documented. It is also rec-
should be defined and documented in the laboratory.
ommended that the test documentation should include
Errors or incorrect responses for proficiency specimens
pre-hybridization and hybridization conditions and
are documented, along with the corrective action taken,
probe and hybridization buffer lot numbers.
if necessary.
In situ results, such as FISH, are correlated with histo-
logical findings (stained sections) of tissue morphology.
DOCUMENTATION OF TEST RESULTS This is required when the molecular target detection is
significant in specific cells, for example, with p53 detec-
Test results in the form of electropherograms, gel tion in tumor cells. Documentation includes images of
images, and autoradiograms should be of a sufficiently at least one normal cell with at least two abnormal cell
high quality that results are unequivocal. This includes results. These images are cross-referenced or retained
together with photographs, films, and autoradiographs known sequence changes is sometimes performed only
generated from additional testing of the same specimen. on one template strand; however, sequencing of both
All these records are labeled with patient identification, strands is best.
sample numbers, run identifiers, and the date of assay. The criteria for the acceptance of sequencing data
All of the raw data are retained with the final report include correct assignment of the nucleotide sequence in
and clinical interpretation of the test results. Careful a defined region surrounding the critical area, not includ-
documentation is important because molecular diagnosing the amplification primer-binding sites. Furthermore,
tic results may differ from results from other laboratories a specified level of band or peak quality (intensity or
or from the clinical diagnosis. Such discrepancies occur fluorescence levels, respectively) with a reasonably low
most often with amplification methods because of their background is assigned. Defined limits of fluorescence
high sensitivity. If a molecular result is questioned, inves- ratios are set to identify true heterozygous base positions.
tigation of the discrepancy includes a review of the raw Ideally, a heterozygous position will have equal fluores-
data. The results of this investigation, along with any cor- cence contribution from the two genotypes, and the peak
rective action taken, are noted in the laboratory records. height will be approximately half that of a homozygous
genotype at that position. Results are expressed in the
standard nomenclature for DNA or protein sequences
Gene Nomenclature
(see Chapter 9).
Reporting of results from genetic testing requires The utility of sequence data requires published
unequivocal identification of the gene being tested. normal or type-specific sequences. In the case of gene
Gene names are sometimes not consistent. There can mutations, electronic or published databases of known
be multiple names referring to the same gene. For mutations and polymorphisms are available for fre-
example, the official HUGO Gene Nomenclature Com- quently tested genes. These records, especially Inter-
mittee (HGNC) gene name ERBB2 is also called HER2, net databases, are updated regularly. Newly discovered
HER-2, and EGFR2. Even the official name can be mutations are classified according to the type of muta-
changed by the HGNC. Ongoing efforts to implement tion; the laboratory director or consultant uses published
the universal usage of standard official gene names and guidelines to determine if the mutation is clinically sig-
symbols by molecular laboratory professionals are docu- nificant.24 For example, a silent mutation will not affect
mented at http://www.genenames.org. Meanwhile, labo- protein function, whereas a frameshift mutation will.
ratory reports may refer to “also known as” names for
the same gene, for example, ERBB2 (HER2). Gene name
aliases can be found at the National Center for Biologi- REPORTING RESULTS
cal Information (NCBI) Gene Database (http://www
.ncbi.nlm.nih.gov/gene/). It is most important to use the Test results are reported in terms that are unambigu-
gene name or symbol recognized by the laboratory’s ous to readers who may not be familiar with molecu-
medical community. lar methods or terminology. The test report must clearly
convey the method or manufactured kit used, the locus,
the mutation or organism tested, the analytical interpre-
Gene Sequencing Results
tation of the raw data, and the clinical interpretation of
Direct sequencing is increasingly used in clinical appli- the analytical result. This interpretation includes the pen-
cations to detect gene mutations or to type microorgan- etrance of mutations, that is, the probability of having a
isms. Sequence data must be of adequate quality with mutation but not getting the associated disease.
acceptably low baseline, especially if heterozygous The likelihood of false-positive or false-negative
target states are to be detected. Each nucleotide peak results is also included in a report. Mutation detection is
or band should be unequivocal. Sequencing should be not guaranteed, especially in large genes with hundreds
performed on both complementary strands of the tem- of possible mutations that may or may not effectively
plate to confirm sequenced mutation or type. Repeated compromise gene function. The mutation detection rate
sequencing across the same area, or resequencing, for for this type of gene and the residual risk of undetected
mutations are therefore included in the test report. Nega- telecopy transmission contain confidential information
tive results from tests for specific point or chromosomal belonging to the sender that is legally privileged. This
mutations are reported in terms of the sensitivity of information is intended only for the use of the individ-
the test (e.g., less than 0.01% chance of mutation) or, ual or entity named above. The authorized recipient of
alternatively, accompanied with the sensitivity levels of this information is prohibited from disclosing this infor-
the test. For parentage reports, the combined paternity mation to any other party and is required to destroy the
index, the probability of paternity as a percentage, the information after its stated need has been fulfilled. If
prior probability of paternity used in calculations, and you are not the intended recipient, you are hereby noti-
the population used for comparison are reported. fied that any disclosure, copying, distributing, or action
The laboratory director, pathologist, or other clinical taken in reliance on the contents of these documents is
expert reviews the analytical interpretation, determines strictly prohibited. If you have received this telecopy in
the clinical interpretation, and verifies the final results error, please notify the sender immediately to arrange
with an actual or electronic signature on the test report. for return or destruction of these documents.”
An internal laboratory summary sheet or database is Test results are not released to employers, insurers,
often useful for compiling pertinent information. Test or other family members without the patient’s expressed
results should not be released before they are reviewed consent. Any data discussed in a public forum are pre-
by the director. Molecular diagnostic tests, in particu- sented such that no patient or pedigree is identifiable by
lar, may have technical complexities that influence the the patient or the general audience. Written consent from
meaning of the test result. These results are best com- the patient may be required under some circumstances.
municated with the clinical significance of the laboratory Each institution will have a department that oversees the
findings. lawful use of confidential information.
When class I ASRs are used in an analytical method, Technologists working in the area of molecular
the following disclaimer is included in the test report: pathology will encounter tests in which the final details
“This test was developed, and its performance charac- are determined empirically. As a result, a test proce-
teristics determined by (laboratory name). It has not dure may differ from one laboratory to another. Even
been cleared or approved by the U.S. Food and Drug after the test procedure is established, troubleshooting is
Administration.” Additional language is also recom- sometimes required as the procedure is used on a routine
mended by the CAP: “The FDA does not require this basis. Some reactions that work well for short-term
test to go through premarket FDA review. This test is research may prove to be less consistent and reproduc-
used for clinical purposes. It should not be regarded as ible than is required in the clinical laboratory setting.
investigational or for research. This laboratory is certi- Biotechnology is quickly developing standard reagent
fied under the Clinical Laboratory Improvement Amend- sets and instrumentation for the most popular tests, but
ments (CLIA) as qualified to perform high complexity these also differ from one supplier to another. Further-
clinical laboratory testing.”25 more, due to market demands, test reagent kits may be
The disclaimer is not required for tests using reagents modified or discontinued. If replacement reagents are
that are sold together with other materials or an instru- available, they may not be identical to those previously
ment as a reagent set nor for reagents sold with instruc- used. Ongoing tests then must be optimized. This can be
tions for use. a concern where turnaround times are critical.
Maintaining the confidentiality of molecular test It then becomes the responsibility of the technolo-
results is essential. All results, and particularly molecu- gist to perform and monitor tests on a regular basis to
lar genetic results, may affect insurability, employment, maintain the consistency and accuracy of results. The
or other family members. Results are released only to technologist who understands the biochemistry and
the ordering physician or other authorized personnel molecular biology of these tests will be better able to
such as genetic counselors or nurse coordinators. Tech- respond to these problems. In addition, with the quick-
nologists should refer requests for patient data to super- ened evolution of the sciences, a knowledgeable tech-
visors. Data sent by facsimile must be accompanied by a nologist can better recognize significant discoveries that
disclaimer such as: “The documents accompanying this offer the potential for test improvement.
4. Tanner M, Berk LS, Felten DL, Blidy AD, Bit SL, Ruff DW. Sub-
STUDY QUESTIONS stantial changes in gene expression level due to the storage tem-
perature and storage duration of human whole blood. Clinical and
Laboratory Haematology 2002;24:337–341.
What actions should be taken in the following 5. Chai V, Vassilakos A, Lee Y, Wright JA, Young AH. Optimization
situations? of the PAXgeneTM blood RNA extraction system for gene expres-
sion analysis of clinical samples. Journal of Clinical Laboratory
1. An unlabeled collection tube with a requisition for Analysis 2005;19:182–188.
a factor V Leiden test is received in the laboratory. 6. Duale N, Lipkin WI, Briese T, Aarem J, Rønningen KS, Aas KK,
Magnus P, Harbak K, Susser E, Brunborg G. Long-term storage of
blood RNA collected in RNA stabilizing Tempus tubes in a large
2. After PCR, the amplification control has failed to biobank—evaluation of RNA quality and stability. BMC Research
yield a product. Notes 2014;7:633.
7. Lou J, Mirsadraei L, Sanchez DE, Wilson RW, Shabihkhani M,
3. An isolated DNA sample is to be stored for at least Lucey GM, Wei B, Singer EJ, Mareninov S, Yong WH. A review
of room temperature storage of biospecimen tissue and nucleic
6 months.
acids for anatomic pathology laboratories and biorepositories.
Clinical Biochemistry 2014;47:267–273.
4. A bone marrow specimen arrives at the end of 8. Takeo S, Ishii A, Segawa T, Takagi Y, Kobayashi Y, Itou T.
a shift and will not be processed for the Bcl2 Establishing conditions for the storage and elution of rabies
translocation until the next day. virus RNA using FNA® cards. Journal of Veterinary Medicine
2015;77:461–465.
9. Burd E. Validation of laboratory-developed molecular assays for
5. The temperature of a refrigerator set at 8°C (±2°C) infectious diseases. Clinical Microbiological Reviews 2010;23:
reads 14°C. 550–576.
10. Pyne M, Konnick EQ, Phansalkar A, Hillyard DR. Evaluation of
6. A PCR test for the BCR/ABL translocation was the Abbott investigational use only RealTime hepatitis C virus
(HCV) assay and comparison to the Roche TaqMan HCV analyte-
negative for the patient sample and for the
specific reagent assay. Journal of Clinical Microbiology 2009;47:
sensitivity control. 2872–2878.
11. Goedel S, Rullkoetter M, Weisshaar S, Mietag C, Leying H,
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9. The expiration date on a reagent has passed. pathology tests. Archives of Pathology & Laboratory Medicine
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13. De Vore K, Agrwal Y, Alspach TD, Budd JR, Durazo-Arvizu
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Appendix A
Study Question Answers
Chapter 1 Study Question Answers Restriction Enzyme Analysis
DNA Structure and Function 1. A plasmid was digested with the enzyme HpaII. On
agarose gel electrophoresis, you observe three bands,
1. What is the function of DNA in the cell? 100, 230, and 500 bp.
Answer: DNA is a storage system for genetic Answer:
information.
a. How many HpaII sites are present in this
2. Compare the structure of the nitrogen bases. How do plasmid? There are three HpaII sites.
purines and pyrimidines differ? b. What are the distances between each site?
100 bp, 230 bp, 500 bp
Answer: Purines have a double ring; pyrimidines c. What is the size of the plasmid? The plasmid is
have a single ring. 830 bp in size.
d. Draw a picture of the plasmid with the
3. Write the complementary sequence to the following:
HpaII sites.
5′AGGTCACGTCTAGCTAGCTAGA3′
Hpall
Answer: 3′TCCAGTGCAGATCGATCGATCT5′ 100 bp
Hpall
4. Which of the ribose carbons participate in the
phosphodiester bond? 500 bp
230 bp
Answer: The 5′ carbon connects to a hydroxyl Hpall
group on the 3′ ribose carbon of the previous
nucleotide. A second cut of the plasmid with BamH1 yields two
pieces, 80 and x bp.
5. Which of the ribose carbons carries the nitrogen
base? e. How many BamH1 sites are in the plasmid?
There are two BamH1 sites.
Answer: The 1′ carbon carries the nitrogen base. f. What is x in base pairs (bp)? 830 bp – 80 bp =
750 bp
6. Why does DNA polymerase require primase
activity? 2. How would you determine where the BamH1 sites
are in relation to the HpaII sites?
Answer: A 3′ hydroxyl group from an existing
nucleotide must be present to form the Answer: Cut the plasmid with both enzymes at
phosphodiester bond. the same time.
473
474 Appendix A • Study Question Answers
3. The plasmid has one EcoR1 site into which you want 2. Underline two inverted repeats in the following RNA.
to clone a blunt-ended fragment. What type of enzyme
Answer: 5′CUGAACUUCAGUCAA
could turn an EcoR1 sticky end into a blunt end?
GCAAAGAGUUUGCACUG3′
Answer: A 5′ to 3′ single-strand exonuclease will
remove the single-stranded overhangs. RNA Processing
Recombination and DNA Transfer 1. Name three processing steps undergone by

messenger RNA.
1. Describe how DNA moves from cell to cell by Answer: mRNA is capped during transcription
(a) conjugation, (b) transduction, and and polyadenylated as part of termination. The
(c) transformation. transcript is further processed by splicing.
Answer:
2. What is the function of polyadenylate polymerase?
a. Cells share DNA by direct cell-to-cell contact.
b. Viral or bacteriophage vectors carry DNA Answer: This enzyme adds adenosine nucleotides
from cell to cell. to the 3′ end of mRNA.
c. Fragmented or plasmid DNA enters cells from
the surrounding environment. 3. What is unusual about the phosphodiester bond
formed between mRNA and its 5′ cap?
2. Which of the three interactions in question 1 would
Answer: The phosphodiester bond involved in
be prevented by a barrier between two mating strains
capping is a 5′–5′ bond, rather than the more
that stops bacterial cells but not smaller particles?
usual 3′–5′ bond.
Answer: Conjugation would be prevented
because there would be no cell-to-cell contact. 4. The parts of the hnRNA that are translated into
Smaller viral particles, plasmids, and fragments protein (open reading frames) are the __exons___.
of DNA would pass through the membrane.
5. The intervening sequences that interrupt protein-
3. After meiosis, gametes produced from diploid coding sequences in hnRNA are ___introns___.
organisms are _haploid_ (haploid/diploid).
Proteins and the Genetic Code
RNA Secondary Structure
1. Indicate whether the following peptides are
1. Draw the secondary structure of the following RNA. hydrophilic or hydrophobic.
The complementary sequences (inverted repeat) are Answer:
underlined.
a. MLWILAA hydrophobic
Answer: 5′CAUGUUCAGCUCAUGUGAAC b. VAIKVLIL hydrophobic
GCU3′ c. CSKEGCPN hydrophilic
d. SSIQKNET hydrophilic
C
U C A
U
e. YAQKFQGRT hydrophilic
G G f. AAPLIWWA hydrophobic
A U g. SLKSSTGGQ hydrophilic
C G
U A 2. Is the following peptide positively or negatively
U A charged at neutral pH?
G C
C A U G C U GWWMNKCHAGHLNGVYYQGGTY
Appendix A • Study Question Answers 475
Answer: The peptide is positively charged with Answer: The 95°C incubation will inactivate
basic side chains pI > pH 7. the protein, preventing its activity in subsequent
steps of the assay.
3. Consider an RNA template made from a 2:1
mixture of C:A. What would be the three amino 9. What is a ribozyme?
acids most frequently incorporated into the protein?
Answer: A ribozyme is an RNA molecule
Answer: Proline (CCA), histidine (CAC), and that can metabolize other molecules like an
threonine would be frequently incorporated. enzyme.
4. What is the peptide sequence encoded in 10. Name the nonprotein prosthetic groups for the
AUAUAUAUAUAUAUA . . . ? following conjugated proteins:
Answer: The protein would alternate isoleucine
(AUA) and tyrosine (UAU). glycoprotein sugars
5. Write the anticodons 5′ to 3′ of the following lipoprotein lipids

amino acids: metalloprotein metal atoms
Answer:
a. L UAA, CAA, AAG, GAG, UAG, CAG
b. T AGU, GGU, UGU, CGU
Chapter 2 Study Questions
c. M CAU Gene Expression
d. H AUG, GUG
e. R ACG, GCG, UCG, CCG 1. Proteins that bind to DNA to control gene expression
f. I AAU, GAU are called trans (or transcription) factors.
6. A protein contains the sequence 2. The binding sites on DNA for proteins that control
LGEKKWCLRVNPKGLDESKDYLSLKSKYLLL. gene expression are __ cis ___ factors.
What is the likely function of this protein? (Note:
See Advanced Concepts box.) 3. How might a single mRNA produce more than one
Answer: This protein has leucine (L) residues protein product?
at sequential seventh positions, forming a Answer: A single RNA may be alternatively
leucine zipper found in transcription factors spliced to produce more than one protein
(DNA-binding proteins). product.
7. A histone-like protein contains the sequence 4. The type of transcription producing RNA that is
PKKGSKKAVTKVQKKDGKKRKRSRK. What continually required and relatively abundant in the
characteristic of this sequence makes it likely to cell is called __constitutive___ transcription.
associate with DNA?
Answer: The positive charge on this protein, 5. A set of structural genes transcribed together on one
facilitating association with the negatively polycistronic mRNA is called an _operon_.
charged DNA, makes it likely to associate
with DNA. The Lac Operon
8. A procedure for digestion of DNA with a restriction Using the depiction of the lac operon in Figure 2.4,
enzyme includes a final incubation step of indicate whether gene expression (transcription)
5 minutes at 95°C. What is the likely purpose of would be on or off under the following conditions:
this final step? (P = promoter; O = operator; R = repressor).
Answer: Answer: CpG islands (more than the expected

frequency of CG dinucleotides within a given
a. P+ O+ R+, no inducer present—OFF
length of DNA), often found 5′ to structural
b. P+ O+ R+, inducer present—ON
genes, are targets for methylation.
c. P– O+ R+, no inducer present—OFF
d. P– O+ R+, inducer present—OFF 5. What is the RISC?
e. P+ O– R+, no inducer present—ON
f. P+ O– R+, inducer present—ON Answer: The RNA-induced silencing complex
g. P+ O+ R–, no inducer present—ON mediates the specific interaction between siRNA
h. P+ O+ R–, inducer present—ON or miRNA and the target RNA.
i. P– O– R+, no inducer present—OFF
j. P– O– R+, inducer present—OFF 6. Name four functions of lncRNAs.
k. P– O+ R–, no inducer present—OFF Answer: lncRNA recruit modifiers of chromatin,
l. P– O+ R–, inducer present—OFF act as decoys that titrate away DNA-binding
m. P+ O– R–, no inducer present—ON proteins such as transcription factors, act as
n. P+ O– R–, inducer present—ON scaffolds to bring two or more proteins into
o. P– O– R–, no inducer present—OFF a complex, or bind to DNA in an enhancer-
p. P– O– R–, inducer present—OFF like control of gene expression from distal cis
elements.
Epigenetics
1. Indicate whether the following events would DNA Quantity/Quality
increase or decrease expression of a gene:
Answer: 1. Calculate the DNA concentration in μg/mL from the
following information:
a. Methylation of cytosine bases 5′ to the gene
would decrease expression. Answer:
b. Histone acetylation close to the gene would a. Absorbance reading at 260 nm from a
increase expression. 1:100 dilution = 0.307
c. siRNAs complementary to the gene transcript
will decrease expression. 0.307 × 50 μg/mL = 15.35 μg/mL
15.35 μg/mL × 100 = 1,535 μg/mL
2. How does the complementarity of siRNA to its b. Absorbance reading at 260 nm from a
target mRNA differ from that of miRNA? 1:50 dilution = 0.307
Answer: The complementarity of siRNA to its 0.307 × 50 μg/mL = 15.35 μg/mL
target is exact. The complementarity of miRNA 15.35 μg/mL × 50 = 767.5 μg/mL
to its target is imperfect.
c. Absorbance reading at 260 nm from a
3. What is imprinting in DNA? 1:100 dilution = 0.172
Answer: DNA imprinting is the marking 0.172 × 50 μg/mL = 8.60 μg/mL
of selected regions of DNA, usually by 8.60 μg/mL × 100 = 860 μg/mL
methylation.
d. Absorbance reading at 260 nm from a
1:100 dilution = 0.088
4. What sequence structures in DNA, usually found
5′ to structural genes, are frequent sites of DNA 0.088 × 50 μg/mL = 4.40 μg/mL
methylation? 4.40 μg/mL × 100 = 440 μg/mL
2. If the volume of the DNA solutions in question 1 generate a signal, whereas single nucleotides will
was 0.5 mL, calculate the yield for (a)–(d). absorb light in spectrophotometry.
Answer:
RNA Quantity/Quality
a. 1,535 μg/mL × 0.50 mL = 767.5 μg
b. 767.5 μg/mL × 0.50 mL = 383.8 μg 1. Calculate the RNA concentration in μg/mL from the
c. 860 μg/mL × 0.50 mL = 430 μg following information:
d. 440 μg/mL × 0.50 mL = 220 μg
Answer:
3. Three DNA preparations have the following A260 and a. Absorbance reading at 260 nm from a
A280 readings: 1:100 dilution = 0.307
For each sample, based on the A260/A280 ratio, is each
preparation suitable for further use? If not, what is 0.307 × 40 μg/mL = 12.28 μg/mL
contaminating the DNA? 12.28 μg/mL × 100 = 1,228 μg/mL
Sample OD260 OD280 b. Absorbance reading at 260 nm from a
(i) 1 0.419 0.230 1:50 dilution = 0.307
0.419/0.230 = 1.82. 0.307 × 40 μg/mL = 12.28 μg/mL
This DNA is suitable for use. 12.28 μg/mL × 50 = 614 μg/mL
(ii) 2 0.258 0.225 c. Absorbance reading at 260 nm from a
0.258/0.225 = 1.15. 1:100 dilution = 0.172
This DNA is not suitable for use due to protein
contamination. 0.172 × 40 μg/mL = 6.88 μg/mL
6.88 μg/mL × 100 = 688 μg/mL
(iii) 3 0.398 0.174
0.398/0.174 = 2.29. d. Absorbance reading at 260 nm from a
This DNA may be suitable for use if RNA does 1:100 dilution = 0.088
not interfere with the subsequent assay. 0.088 × 40 μg/mL = 3.52 μg/mL
4. After agarose gel electrophoresis, a 0.5-microgram 3.52 μg/mL × 100 = 352 μg/mL
aliquot of DNA isolated from a bacterial culture
produced only a faint smear at the bottom of the gel 2. If the volume of the RNA solutions in question 1
lane. Is this an acceptable DNA sample? was 0.5 mL, calculate the yield for (a)–(d).
Answer: This amount of DNA should produce a Answer:

bright band/smear near the top of the gel lane. a. 1,228 μg/mL × 0.50 mL = 614 μg
This DNA is probably degraded and is therefore b. 614 μg/mL × 0.50 mL = 307 μg
unacceptable. c. 688 μg/mL × 0.50 mL = 344 μg
d. 3.52 μg/mL × 0.50 mL = 1.76 μg
5. Compare and contrast the measurement of DNA
concentration by spectrophotometry with analysis by
3. An RNA preparation has the following absorbance
fluorometry with regard to staining requirements and
readings:
accuracy.
A260 = 0.208
Answer: Spectrophotometry requires no DNA
A280 = 0.096
staining. Fluorometry requires staining of DNA to
generate a fluorescent signal. Fluorometry may be Is this RNA preparation satisfactory for use? The
more accurate than spectrophotometry because A260/A280 ratio is 0.208/0.096 = 2.17. This RNA
double-stranded DNA must be intact to stain and preparation is satisfactory for use.
4. A blood sample was held at room temperature for and staining were performed properly. In
5 days before being processed for RNA isolation. this case, the DNA fragments were not loaded
Will this sample likely yield optimal RNA? onto the gel or the method used to produce
the fragments was not successful.
Answer: This sample will not yield optimal
RNA due to degradation and changes in gene
3. How does PFGE separate larger fragments more
expression at room temperature.
efficiently than standard electrophoresis?
5. Name three factors that will affect the yield of RNA Answer: PFGE forces large fragments
from a paraffin-embedded tissue sample. through the gel matrix by repeatedly
Answer: Isolation of RNA from fixed tissue is changing the direction of the current, thus
affected by (1) the type of fixative used, (2) the aligning and realigning the particles with
age/length of storage of the tissue, and (3) the the gel spaces.
preliminary handling of the original specimen.
Other factors such as time of fixation, type of 4. A 6% solution of 19:1 acrylamide:bis-acrylamide is
specimen, and specimen transport conditions will mixed, de-aerated, and poured between glass plates
also affect RNA quality and yield. for gel formation. After an hour, the solution is still
liquid. What might be one explanation for the gel
Chapter 4 Study Questions not polymerizing?
Answer: The nucleating agent and/or
1. You wish to perform an electrophoretic resolution polymerization catalyst were not added to the
of your restriction enzyme–digested DNA. The gel solution.
sizes of the expected fragments range from 100 to
500 bp. You discover two agarose gels polymerizing 5. A gel separation of RNA yields aberrantly
on the bench. One is 0.5% agarose; the other is 2% migrating bands and smears. Suggest two possible
agarose. Which one might you use to resolve your explanations for this observation.
fragments?
Answer: RNA degradation will yield the
Answer: The 2% gel is best for this range of aberrantly migrating bands and smears.
fragment sizes. Improper electrophoresis conditions (buffer,
2. After completion of the electrophoresis of DNA denaturation agent, or gel) will also affect band
fragments along with the proper molecular-weight migration.
standard on an agarose gel, suppose the outcomes
in (a) and (b) were observed. What might be the 6. Why does DNA not resolve well in solution
explanations for each outcome? (without a gel matrix)?
Answer: Answer: Particle movement in solution is
based on the charge/mass ratio. As the mass
a. The gel is blank (no bands, no molecular-weight
of DNA increases, slowing migration, the
standard).
negative charge increases, counteracting the
Because the molecular-weight standard is not
effect of mass.
visible, something is wrong with the general
electrophoresis process. Most likely, staining
7. Why is SYBR green less toxic than EtBr?
with a DNA-specific dye was omitted.
b. Only the molecular-weight standard is Answer: SYBR green is a minor groove–
visible. binding dye. It does not disrupt the nucleotide
The presence of the molecular-weight sequence of DNA. Ethidium bromide (EtBr) is
standard indicates that the electrophoresis an intercalating agent that slides in between the
nucleotide bases in the DNA, which can cause d. CATCGCGATCTGCAATTACGACGATAA

changes in the nucleotide sequence (mutations) GTAGCGCTAGACGTTAATGCTGCTATT
in the DNA.
Answer: (15 × 2°C) + (12 × 4°C) = 78°C
8. What are the general components of loading buffer 2. What is the purpose of denaturation of a double-
used for introducing DNA samples to submarine stranded target DNA after electrophoresis and prior
gels? to transfer in a Southern blot?
Answer: Gel loading buffers contain a density Answer: Double-stranded target DNA is
agent (Ficoll, glycerol, sucrose) to facilitate denatured to allow hybridization of the
placing the liquid sample in the gel well probe. Also, single-stranded DNA binds to the
underneath the buffer surface and a tracking nitrocellulose fibers in the blotting membrane.
dye to follow the migration of the sample during
electrophoresis. 3. Name two ways to permanently bind nucleic acid
to nitrocellulose following transfer.
9. Name two dyes that are used to monitor
the migration of nucleic acid during Answer: To permanently bind the nucleic
electrophoresis. acid, bake the filter at 80°C for 30 minutes.
Alternatively, nucleic acid will be cross-linked to
Answer: Bromphenol blue and xylene cyanol nitrocellulose upon exposure to ultraviolet light.
green are two of several tracking dyes.
4. If a probe for a Southern blot is dissolved in a
10. When a DNA fragment is resolved by slab gel hybridization buffer that contains 50% formamide,
electrophoresis, a single sharp band is obtained. is the stringency of hybridization higher or lower
What would the equivalent observation be if than if there was no formamide?
this fragment had been fluorescently labeled and
Answer: The stringency is higher because
resolved by capillary electrophoresis?
formamide facilitates denaturation of double-
Answer: Comparable capillary electrophoresis stranded DNA.
results will be a single peak on an
electropherogram. 5. If a high concentration of NaCl was added to a
hybridization solution, how would the stringency
be affected?
Answer: The stringency would be decreased
1. Calculate the melting temperature of the following because high salt concentrations exclude
DNA fragments using the sequences only: nucleic acids, forcing them close together and
promoting hybridization.
a. AGTCTGGGACGGCGCGGCAATCGCA
TCAGACCCTGCCGCGCCGTTAGCGT 6. Does an increase in temperature from 65°C to 75°C
during hybridization raise or lower the stringency?
Answer: (8 × 2°C) + (17 × 4°C) = 84°C
Answer: Increasing the temperature raises the
b. TCAAAAATCGAATATTTGCTTATCTA
stringency. Heat promotes separation of the
AGTTTTTAGCTTATAAACGAATAGAT
hydrogen bonds holding the two single strands
Answer: (20 × 2°C) + (6 × 4°C) = 64°C of double-stranded DNA together.
c. AGCTAAGCATCGAATTGGCCATCGTGTG
7. At the end of the Southern blot procedure, what
TCGATTCGTAGCTTAACCGGTAGCACAC
would the autoradiogram show if the stringency
Answer: (14 × 2°C) + (14 × 4°C) = 84°C was too high?
Answer: If the stringency was too high, CGH arrays detect deletions and amplifications
faint or no bands will be present on the of chromosomal regions, whereas expression
autoradiogram. arrays detect changes in the RNA levels of
specific genes.
8. A Northern blot is performed on an RNA transcript
with the sequence GUAGGUATGUAUUUGGGCG
CGAACGCAAAA. The probe sequence is Chapter 6 Study Questions
GUAGGUATGUAUUUGGGCGCG. Will this
probe hybridize to the target transcript? 1. A master mix of all components (except template)
necessary for PCR contains what basic
Answer: This probe will not bind to the target ingredients?
transcript. The sequences are identical and not
complementary. Because RNA does not have Answer: An example master mix (without
a complementary partner, the probe must be template) will contain the following:
complementary to the transcript sequence. primers
dATP, dCTP, dGTP, dTTP
9. In an array CGH experiment, three test samples KCl or other monovalent cation
were hybridized to three microarray chips. Tris buffer
Each chip was spotted with eight gene probes MgCl2
(Genes A–H ). The following table shows the polymerase
results of this assay expressed as the ratio of test
DNA to reference DNA. Are any of the eight 2. The final concentration of Taq polymerase is to be
genes consistently deleted or amplified in the test 0.01 units/μL in a 50-μL PCR. If the enzyme is
samples? If so, which ones? supplied at 5 units/μL, how much enzyme would
you add to the reaction?
Gene Sample 1 Sample 2 Sample 3 a. 1 μL
b. 1 μL of a 1:10 dilution of Taq
A 1.06 0.99 1.01
c. 5 μL of a 1:10 dilution of Taq
B 0.45 0.55 0.43 d. 2 μL
C 1.01 1.05 1.06 Answer: Using ratio and proportion to achieve
the concentration of 0.01 units/μL, the enzyme
D 0.98 1.00 0.97
should be 0.01/5.00 = x/50. Because x = 0.1,
E 1.55 1.47 1.62 1 μL of a 1:10 dilution of Taq (b) is the correct
answer.
F 0.98 1.06 1.01
G 1.00 0.99 0.99 3. Primer dimers result from

H 1.08 1.09 0.90 a. high primer concentrations.
b. low primer concentrations.
Answer: Gene B is consistently deleted (test/ c. high GC content in the primer sequences.
reference < 1.0), and gene E is consistently d. 3′ complementarity in the primer
amplified (test/reference > 1.0). sequences.
Answer: Primer dimers result from 3′
10. What are two differences between CGH arrays and
complementarity in the primer sequences
expression arrays?
(d). Without this “self-priming” of primers,
Answer: CGH arrays are performed on DNA, concentration and GC content will not generate
whereas expression arrays use RNA. PCR products.
4. Which control is run to detect contamination? 7. How many copies of a target are made after 30
cycles of PCR?
a. Negative control
b. Positive control a. 2 × 30
c. Molecular-weight marker b. 230
d. Reagent blank c. 302
d. 30/2
Answer: The reaction mix without template,
which is the reagent blank (d), is included Answer: Assuming 100% efficiency, there
to detect contamination. Negative and should be 230 (b) copies of the target after
positive controls monitor the performance 30 PCR cycles. This reflects doubling of the
and accuracy of the reaction. The molecular- amplifiable template with each cycle.
weight marker allows assessment of the
PCR product size and monitors the gel 8. What are the three steps of a standard PCR cycle?
performance.
Answer: A standard three-step PCR cycle
includes a denaturation step where the double-
5. Nonspecific extra PCR products can result from stranded template becomes single-stranded for
a. mispriming. binding of primers in the second or annealing
b. high annealing temperatures. step of the cycle. Extension or addition of
c. high agarose gel concentrations. nucleotides to the 3′ ends of the primers is the
d. omission of MgCl2 from the PCR. third step, resulting in two double-stranded
copies of the original template.
Answer: Nonspecific products result from
mispriming (a). High annealing temperatures
9. Which of the following is a method for purifying a
and omission of magnesium from the reaction
PCR product?
would lessen the production of amplicons. The
agarose gel concentration is not related to the a. Treating with uracil N glycosylase
PCR reaction. b. Adding divalent cations
c. Putting the reaction mix through a spin
column
6. Using which of the following is an appropriate way
d. Adding DEPC
to avoid PCR contamination?
Answer: Post-PCR steps of protocols may
a. High-fidelity polymerase
be affected by residual materials and buffer
b. Hot-start PCR
components from the PCR reaction. Putting
c. A separate area for PCR setup
the reaction mix through a spin column (c) will
d. Fewer PCR cycles
collect the PCR product, which can be eluted
Answer: A separate pre-PCR setup area (c) in a purer solution. N glycosylase will digest
with separate equipment and reagents is an DNA containing uracil, which can purify non–
appropriate way to prevent contamination. uracil-containing DNA, but this mix of DNAs is
High-fidelity polymerase will copy the unlikely to occur in the PCR product. Divalent
template with minimal PCR sequence cations are necessary for polymerase enzyme
artifacts but cannot distinguish contamination activity to produce the PCR product but not
from the desired template. Hot-start PCR can purify it. DEPC is an agent that prevents
inhibit mispriming but not contamination. Using RNase from digesting RNA. Although RNA
fewer PCR cycles will lower the amount of is not a component product of a PCR
final product but does not prevent misprimed reaction, if present, it would not be removed
amplification. by DEPC.
10. In contrast to standard PCR, real-time PCR is Answer: There are multiple primers that can
satisfy these requirements. One primer pair
a. quantitative.
example is given.
b. qualitative.
c. labor-intensive. 5′TATTTAGTTA TGGCCTATAC ACTATTTGTG
d. sensitive. AGCAAAGGTG ATCGTTTTCT GTTTGAGATT
TTTATCTCTT GATTCTTCAA AAGCATTCTG
Answer: Real-time PCR (qPCR) is quantitative
AGAAGGTGAG ATAAGCCCTG AGTCTCAGCT
(a). It is not labor-intensive compared
ACCTAAGAAA AACCTGGATG TCACTGGCCA
with standard PCR because no post-PCR
CTGAGGAGC T TTGTT TCAAC
electrophoresis is required to see the product.
CAAGTCATGT> GCATTTCCAC
qPCR can be very sensitive but not necessarily
more so than standard PCR. GTCAACAGAA TTGTTTATTG TGACAGTTAT
ATCTGTTGTC CCTTTGACCT TGTTTCTTGA
11. In real-time PCR, fluorescence is not generated by
which of the following? AGGTTTCCTC GTCCCTGGG C AATTCCGCAT
TTAATTCAT G GTATTCAGGA
a. FRET probes
b. TaqMan probes <G TTAAGGCGTAAATTAAGTA
c. SYBR green TTACATGCAT GTTTGGTTA AACCCATGAGA
d. Tth polymerase TTCATTCAGT TAAAAATCCA
Answer: Tth polymerase (d) is a DNA GATGGCGAAT3′
replication enzyme and does not produce a Design one set of primers (forward and reverse) to gen-
signal. Probes and fluorescent dyes are used for erate an amplicon containing the underlined base.
product detection in qPCR.
The primers should be 20 bases long.
12. Prepare a table that compares PCR, LCR, bDNA, The amplicon must be 100 to 150 bp in size.
TMA, Qβ replicase, and hybrid capture with regard The primers must have similar melting temperatures
to the type of amplification, target nucleic acid, (Tm), +/– 2°C.
type of amplicon, and major enzyme(s) for each. The primers should have no homology in the last
three 3′ bases.
Target (DNA Amplified a. Write the primer sequences 5′→3′ as you
Amp Method or RNA) Product would if you were to order them from the DNA
PCR, LAMP, DNA, RNA Target (DNA)
synthesis facility.
HDA, RMA Answer: There are multiple answers to this
TMA (NASBA) RNA, DNA Target (RNA) question. One example:
Qβ replicase DNA Probe (DNA, RNA) Forward primer: 5′TTGTT

TCAACCAAGTCATGT 3′
LCR DNA Probe (DNA)
Reverse primer:
bDNA DNA, RNA Signal 5′ATGAATTAAATGCGGAATTG3′
Hybrid Capture DNA, RNA Signal b. Write the Tm for each primer that you have
designed.
13. Examine the following sequence. (The Answer: Forward primer:
complementary strand is not shown.) You (13 × 2°C) + (7 × 4°C) = 54°C
are devising a test to detect a mutation at the
underlined position. Reverse primer: (14 × 2°C) + (6 × 4°C) = 52°C
(Ideally, forward and reverse primer pairs 4. Write the numerical and structural chromosomal
should have similar annealing temperatures abnormalities represented by the following
±2°C.) genotypes:
14. How does nested PCR differ from multiplex PCR? 47,XY, +18 trisomy 18
46,XY, del(16) deletion in the short arm of
Answer: Nested PCR is done in two rounds;
p(14) chromosome 16 at region 1,
that is, the PCR product of round one is used as
band 4
the template for round two. Multiplex PCR uses
iso(Xq) isochromosome formed
more than one primer pair in a single round of
by centromeric joining of
PCR.
two long arms of the X
chromosome
15. What replaces heat denaturation in strand
46,XX del(22) deletion in the long arm of
displacement amplification?
q(11.2) chromosome 22 at region 1,
Answer: Enzymatic nicking of the double- band 1, subband 2
stranded probe product produces a 3′ end for 45,X loss of one X or the
copying the uncut strand while displacing its Y chromosome
complement.
5. A chromosome with a centromere located such
that one arm of the chromosome is longer than the
Chapter 7 Study Questions other arm is called
a. metacentric.
1. What chromosomal location is indicated by
b. paracentric.
15q21.1?
c. telocentric.
Answer: This location is on the long arm d. submetacentric.
of chromosome 15, region 2, band 1,
Answer: A chromosome with a long and short
subband 1.
arm is submetacentric (d). In a metacentric
chromosome, the centromere is near the middle
2. During interphase FISH analysis for the t(9;22)
so that the arms are of approximately equal
translocation, one nucleus was observed with two
length. One arm is very short in telocentric
normal signals (one red for chromosome 22 and
chromosomes where the centromere is close
one green for chromosome 9) and one composite
to one end. Paracentric refers to an inversion
red/green signal. Five hundred other nuclei
within one arm of a chromosome, not involving
were normal. What is one explanation for this
the centromere.
observation?
6. A small portion from the end of chromosome 2
Answer: The composite signal indicates
has been found on the end of chromosome 15,
the presence of a translocation between
replacing the end of chromosome 15, which has
chromosomes 9 and 22; however, because only
moved to the end of chromosome 2. This mutation
1 of 500 nuclei showed the composite signal,
is called a(n)
the more likely explanation is that this is an
artifactual result, possibly due to the overlap of a. reciprocal translocation.
chromosomes on the slide. b. inversion.
c. deletion.
3. Is 47;XYY a normal karyotype? d. robertsonian translocation.
Answer: No. This karyotype is aneuploid with Answer: The exchange of portions of
polysomy Y (one extra Y chromosome). chromosomes with no loss of genetic material
is a reciprocal translocation (a). An inversion fixed onto a slide, treated with trypsin, and then
reverses the orientation of DNA, only involving stained with Giemsa. The resulting banding pattern
one chromosome. Deletions are a loss of is called
material, from 1 bp to millions of bp from
a. G banding.
chromosomes. In robertsonian translocations,
b. Q banding.
chromosomes break at their centromeres, and
c. R banding.
the long arms fuse to form a single chromosome
d. C banding.
with one centromere.
Answer: Giemsa staining produces G banding
7. Phytohemagglutinin is added to a cell culture when
(a). When chromosomes are stained with the
preparing cells for karyotyping. The purpose of the
fluorescent dyes quinacrine and quinacrine
phytohemagglutinin treatment is to
mustard, the resulting fluorescence pattern
a. arrest the cell in metaphase. visualized after staining is Q banding.
b. spread out the chromosomes. Treatment of chromosomes with acridine
c. fix the chromosomes on the slide. orange dye will produce a pattern opposite to
d. stimulate mitosis in the cells. the G banding pattern called R banding. Alkali
treatment of chromosomes results in centromere
Answer: Cells must enter metaphase for
staining, or C banding.
karyotyping. Mitosis is stimulated in culture
using phytohemagglutinin (d). Once the cells 10. A FISH test with a centromere 13 probe is ordered
go into the cell cycle, Colcemid is used to for a suspected case of Patau syndrome (trisomy
arrest the cells in metaphase. Chromosomes 13). How many signals per nucleus will result if
are distributed from the nucleus by lysis with the test is positive for Patau syndrome?
hypotonic buffer. Chromosomes are fixed on the
slide with methanol. Answer: The FISH results will reveal three signals
per nucleus with a probe to chromosome 13.
8. A centromeric probe is used to visualize
chromosome 21. Three fluorescent signals are 11. What would be the results if a centromere
observed in the cell nuclei when stained with 13 probe was used on a case of Edward syndrome
this probe. These results would be interpreted as (trisomy 18)?
consistent with
Answer: The FISH results will reveal two
a. a normal karyotype. signals per nucleus with a probe to chromosome
b. Down syndrome. 13. A probe to centromere 18 would yield three
c. Klinefelter syndrome. signals per nucleus.
d. technical error.
Answer: Three centromeric signals instead of 12. Angelman syndrome is caused by a microdeletion
two from chromosome 21 is a finding consistent in chromosome 15. Which method, karyotyping or
with Down syndrome (trisomy 21; b). The metaphase FISH, is better for accurate detection of
presence of two centromeric signals for each this abnormality? Why?
chromosome is normal. Klinefelter syndrome Answer: Metaphase FISH is preferred
is indicated by an extra X chromosome in men over karyotyping for the detection of
(47;XXY). Technical error can result in aberrant microdeletions. The lower resolution of
signals, such as chromosome overlap; however, karyotyping makes the detection of small
these artifactual signals are usually rare. deletions difficult.
9. Cells were harvested from a patient’s blood, 13. The results of a CGH analysis of Cy3 (green)-
cultured to obtain chromosomes in metaphase, labeled test DNA with Cy5 (red)-labeled reference
DNA on a normal chromosome spread revealed 3. On a size-exclusion column, large molecules will
a bright red signal along the short arm of elute___before_____ (before/after) small molecules.
chromosome 3. How is this interpreted?
4. MALDI methods separate ions by
a. 3p deletion
b. 3q deletion a. molecular volume.
c. 3p amplification b. mass.
d. 3q amplification c. charge.
d. mass and charge.
Answer: The red signal from the reference
chromosome region indicates a loss or deletion Answer: MALDI methods separate ions by
in the test chromosome at 3p (a). Amplification mass and charge (d), regardless of volume.
would yield a green signal at this location. Molecules to be assessed are ionized before they
Events at 3p would not affect the signal at 3q. are attracted to travel through a magnetic field.
Low-mass ions and more highly charged ions
14. A break-apart probe is used to detect a translocation. move faster through the drift space than ions
The results of FISH analysis show two signals in with higher mass and lower charge. Thus, the
70% of the nuclei counted and three signals in 30% time of ion flight differs according to the mass-
of the nuclei. Is there a translocation present? to-charge ratio (m/z).
Answer: A translocation is present, indicated 5. What is a heteroduplex?
by the nuclei in which three signals appear.
The probe spans the translocation breakpoint, Answer: A heteroduplex is one double-
producing two separate signals when a stranded DNA molecule with one or more
translocation occurs. The third signal is the noncomplementary bases.
intact homologous chromosome. 6. Exon 4 of the HFE gene from a patient suspected
to have hereditary hemochromatosis was amplified
15. What FISH technique is most useful for the
by PCR. The G to A mutation, frequently found
detection of multiple complex genomic mutations?
in hemochromatosis, creates an Rsa1 site in exon
Answer: Spectral karyotyping labels each 4. When the PCR products are digested with
chromosome with a different fluorescent color Rsa1, what results (how many bands) would you
so that multiple complex genomic mutations are expect to see if the patient has the mutation if no
more clearly identified. other Rsa1 sites are naturally present in the PCR
product?
Chapter 8 Study Questions Answer: Digestion with RsaI would produce
two bands if the patient has the mutation and if
1. What characteristic of the genetic code facilitates no other RsaI sites are naturally present in the
identification of open reading frames in DNA PCR product.
sequences?
7. Which of the following methods identifies the
Answer: Out-of-frame or chance consecutive codons
presence of a mutation but not the mutant sequence?
tend to be short, often ending in a stop codon.
a. SSP-PCR
2. Compare and contrast EIA with Western blots for b. SSCP
the detection of protein targets. c. PCR-RFLP
d. NGS
Answer: The EIA method involves liquid
handling and is more easily automated and Answer: SSCP screens for mutations by
analyzed than the Western blot. changes in conformers so that the presence
of a mutation is detected but not the mutant 2. After an automated dye primer sequencing run, the
sequence (b). SSP-PCR relies on primers electropherogram displays consecutive peaks of the
designed to bind the mutant base, and RFLP following colors:
utilizes restriction enzymes with recognition red, red, black, green, green, blue, black, red,
sites containing the potentially mutated base. green, black, blue, blue, blue
In both cases, the mutant sequence is known. If the computer software displays the fluors from
NGS allows detection of many variant bases in a ddATP as green, ddCTP as blue, ddGTP as black,
sequence context. and ddTTP as red, what is the sequence of the
region given?
8. What is the effect on the protein when a codon
sequence is changed from TCT to TCC? Answer: Based on the peak colors, the sequence
is 5′TTGAACGTAGCCC3′.
Answer: There would be no effect on the codon
sequence because TCT and TCC both code for 3. A dideoxy sequencing electropherogram displays
the same amino acid, serine. Codon usage may, bright (high, wide) peaks of fluorescence,
however, affect translation efficiency. obliterating some of the sequencing peaks. What
is the most likely cause of this observation? How
9. A reference sequence, ATGCCCTCTGGC, might it be corrected?
is mutated in malignant cells. The following
Answer: The likely cause is the presence of
mutations in this sequence have been described.
unincorporated labeled dideoxynucleotides or
Express these mutations using the accepted gene
dye blobs. Cleaning the DNA ladder with spin
nomenclature (A = nucleotide position 1).
columns, ethanol precipitation, or bead binding
ATGCGCTCTGGC 5C>G will correct this problem.
ATGCCCTC - -GC 9_10del or 9_10delTG
ATAGCCTCTGGC 3_4delGCinsAG or 4. In a manual sequencing reaction, the sequencing
3_4delinsAG ladder on the polyacrylamide gel is very bright
ATGTCTCCCGGC 4_9inv6 and readable at the bottom of the gel, but the
larger (slower-migrating) fragments higher up are
10. A reference peptide, MPSGCWR, is subject very faint. What is the most likely cause of this
to inherited alterations. The following peptide observation? How might it be corrected?
sequences have been reported. Express these Answer: The loss of longer products is
mutations using the accepted nomenclature caused by an overly high dideoxynucleotide/
(M = amino acid position 1). deoxynucleotide ratio. The problem can be
MPSTGCWR s3_g4inst or s3_g4ins1 corrected by lowering the concentrations of
MPSGX c5x dideoxynucleotides in the sequencing reaction.
MPSGCWLVTGX r7inslvtgx or r7ins5 or r7*
5. In an analysis of the TP53 gene for mutations,
or r7lfsx5
the following sequences were produced. For each
MPSGR c5rdel2
sequence, write the expected sequence of the
MPSGCWGCWR w6_r7insgcw or w6_r7ins3
opposite strand that would confirm the presence of
the mutations detected.
Chapter 9 Study Questions Answer:
5′TATCTGTTCACTTGTGCCCT3′ (Normal)
1. Read 5′ to 3′ the first 15 bases of the sequence in
5′TATCTGTTCATTTGTGCCCT3′ (Homozygous
the gel on the right in Figure 9.7 (p. 229).
substitution)
Answer: The gel sequence is read 5′AGGGCACAAATGAACAGATA3′
from the bottom of the gel to the top: 5′TATCTGT(T/G)CACTTGTGCCCT3′
5′ATCGTCCCTAAGTCA3′ (Heterozygous substitution)
5′AGGGCACAAGTG(C/A)ACAGATA3′ 10. Which of the following projects would require

5′TATCTGTT(C/A)(A/C)(C/T)T(T/G)(G/T)(T/G) next-generation sequencing?
(G/C)CC(C/T)(T/… 3′) (Heterozygous deletion)
a. Mapping a mutation in the hemochromatosis
5′… /A)(A/G)GG(G/C)(C/A)(A/C)(C/A)A(A/G)
gene
(G/T)(T/G)AACAGATA3′
b. Sequencing a viral genome
c. Characterizing a diverse microbial
6. A sequence, TTGCTGCGCTAAA, may be
population
methylated at one or more of the cytosine residues.
d. Typing a single bacterial colony
After bisulfite sequencing, the following results are
obtained: Answer: Characterizing a diverse microbial
population (c) would require high-throughput
Bisulfite treated: TTGUTGCGUTAAA
(massive parallel) sequencing. Analysis of
Write the sequence showing the methylated
single genes or small genomes can be done
cytosines as CMe.
with standard Sanger sequencing or
Answer: TTGCTGCMeGCTAAA pyrosequencing. Bacterial colony typing can
be performed by MALDI-TOF or biochemical
7. In a pyrosequencing readout, the graph shows methods.
peaks of luminescence corresponding to the
addition of the following nucleotides:
dT peak, dC peak (double height), dT peak, dA peak
What is the sequence? Chapter 10 Study Questions
Answer: Based on the peak order and heights,
1. Consider the following STR analysis.
the sequence is TCCTA.
8. Why is it necessary to add adenosine residues Locus Child Mother AF1 AF2
in vitro to ribosomal RNA before capture for
sequencing? D3S1358 15/ 15 15 15 15/16
Answer: mRNA naturally has a polyA 3′ vWA 17/ 18 17 17/18 18

terminus required for immobilization by polyT
FGA 23/ 24 22/23 20 24
hybridization. RNA species without a polyA tail
must be treated with polyA polymerase to add TH01 6/ 10 6/7 6/7 9/10
the 3′ polyA tail.
TPOX 11/ 11 9/11 9/11 10/11
9. Which of the following is next-generation CSF1PO 12/ 12 11/12 11/13 11/12
sequencing?
D5S818 10/ 12 10 11/12 12
a. Maxam–Gilbert
b. Tiled microarray D13S317 9 /10 10/11 10/11 9/11
c. Dideoxynucleotide chain terminator sequencing
d. Reversible dye terminator sequencing a. Circle the child’s alleles that are inherited from
the father.
Answer: Reversible dye terminator (d) is
one example of NGS technology. Maxam– Answer: (See table.)
Gilbert sequencing and tiled arrays are not
b. Which alleged father (AF) is not excluded as
used in clinical sequencing applications.
the biological parent?
Dideoxynucleotide chain termination sequencing
(Sanger sequencing) technology is used for Answer: Based on the circled alleles, AF2 is not
lower-throughput sequencing protocols. excluded.
2. The following evidence was collected for a Answer: For the shared alleles (underlined), the
criminal investigation. combined paternity index is:
Locus Victim Evidence Suspect 0.853 × 2.718 × 1.782 = 4.131
TPOX 11/12 12, 11/12 11

b. With 50% prior odds, what is the probability of
paternity from these three loci?
CSF1PO 10 10 , 9 9/10
Probability of paternity
D13S317 8/10 10, 8/10 9/12
= (4.131× 0.5) [(4.131× 0.5) + 0.5] = 0.80 = 80%
D5S818 9/11 10/11, 9/11 11
Answer: At least 8 loci are commonly tested for
TH01 6/10 6/10 , 8/10 5/11 paternity.
FGA 20 20 , 20/22 20
vWA 15/17 18, 15/17 15/18 4. Consider the following theoretical allele
frequencies for the loci indicated.
D3S1358 14 15/17, 14 11/12
Locus Alleles Allele Frequency

The suspect is heterozygous at the amelogenin
locus. CSF1PO 14 0.332
a. Is the suspect male or female? D13S317 9,10 0.210, 0.595
Answer: The suspect is male (XY), as indicated TPOX 8,11 0.489, 0.237
by heterozygosity at the amelogenin locus.
b. In the evidence column, circle the alleles a. What is the overall allele frequency for this
belonging to the victim. genotype, using the product rule?
Answer: (See table.) Answer: The allele frequency = 0.332 ×

0.332 × 0.21 × 0.595 × 0.489 × 0.237 =
c. Should the suspect be held or released? 0.001596
Answer: The genotype of the suspect is not b. What is the probability that this DNA
consistent with the remaining (un-circled) found at two sources came from the same
alleles. The suspect should be released. person?
3. A child and an alleged father (AF) share alleles Answer: 1/0.001596 = 626.5. It is 626 times
with the following paternity index. more likely that the two DNA samples
came from the same person as from
two random persons in the population.
Paternity Index Legal identity (CODIS) is based on
Locus Child AF for Shared Allele 12 core loci plus amelogenin with recent
recommendations for additional loci being
D5S818 9,10 9 0.853
added.
D8S1179 11 11,12 2.718
D16S539 13,14 10,14 1.782 5. STR at several loci were screened by capillary
electrophoresis and fluorescent detection for
a. What is the combined paternity index from informative peaks prior to a bone marrow transplant.
these three loci? The following results were observed.
9. Which of these would be used for a surname

Locus Donor Alleles Recipient Alleles test: Y-STR, Mini-STR, mitochondrial typing, or
LPL 7,10 7,9 autosomal STR?
F13B 8,14 8
Answer: Y-STR would be used for a surname
or paternal lineage test, going back multiple
FESFPS 10 7 generations. Mini-STR and autosomal STR
are inherited from both parents, making
F13A01 5,11 5,11
inheritance over many generations complex.
Which loci are informative? Mitochondrial alleles are maternally
inherited.
Answer: Loci LPL and FESFPS are informative.
Locus F13B is donor informative. F13A01 is not 10. An ancient bone fragment was found and said
informative. to belong to an ancestor of a famous family.
Living members of the family donated DNA for
6. An engraftment analysis was performed by confirmation of the relationship. What type of
capillary gel electrophoresis and fluorescence analysis would likely be used for this test? Why?
detection. The fluorescence as measured by the Answer: Mitochondrial DNA typing might
instrument under the FESFPS donor peak was be indicated because (1) the small circular,
28,118 units, and that under the FESFPS recipient naturally amplified mitochondrial DNA is more
peak was 72,691. What is the percent donor in this likely to be obtained from the old sample, and
specimen? (2) lineage across several generations can be
Answer: determined using the maternal inheritance of
% Donor = 28,118 (28,118 + 72, 691) = 27.9% mitochondrial type.
11. What is a biological exception to positive

7. The T-cell fraction from the blood sample in
identification by autosomal STR?
question 6 was separated and measured for donor
cells. Analysis of the FESFPS locus in the T-cell Answer: Identical twins (and clones) have
fraction yielded 15,362 fluorescence units under identical nuclear DNA profiles.
the donor peak and 97,885 under the recipient
peak. What does this result predict with regard to 12. A partial STR profile was produced from a highly
T-cell-mediated events such as graft-versus-host degraded sample. Alleles matched to a reference
disease or graft-versus-tumor? sample at five loci. Is this sufficient for a legal
Answer: % Donor = 15,362/(15,362 + 97,885) identification?
= 13.6%. This low percentage of T cells predicts Answer: Five loci are not sufficient for legal
fewer T-cell-mediated events. identification. Nonmatching alleles at any of
these loci may support exclusion.
8. If a child had a Y haplotype including DYS393
allele 12, DYS439 allele 11, DYS445 allele 8, and 13. What is an SNP haplotype? What are tag SNPs?
DYS447 allele 22, what are the predicted Y alleles
Answer: Single-nucleotide polymorphisms
for these loci of the natural father?
(SNPs) that re-inherited together in a block
Answer: The natural father will have the same without combination between them comprise an
Y chromosome as the child: DYS393 allele 12, SNP haplotype. SNP haplotypes are identifiable
DYS439 allele 11, DYS445 allele 8, and DYS447 by two or three representative SNPs (tag SNPs)
allele 22. within the haplotype.
14. Which of the following is an example of linkage 2. Which of the following is a genotypic method
disequilibrium? used to compare two isolates in an epidemiological
investigation?
a. Seventeen members of a population of
1,000 people have a rare disease, and all a. Biotyping
17 people have the same haplotype at a b. Serotyping
particular genetic location on chromosome 3. c. Ribotyping
b. Five hundred people from a population of d. Bacteriophage typing
1,000 people have the same SNP on
Answer: Of the options offered, only ribotyping
chromosome 3.
(c) is genotypic. Biotyping is a phenotypic
Answer: Example (a) is linkage disequilibrium, biochemical reaction. Serotyping is based on
that is, lack of separation of inheritance of the cell surface–antigen phenotype and is used
rare disease and the haplotype. Example (b) is a to classify organisms to the subspecies level.
measure of allele frequency. Bacteriophage typing is based on the variability
between strains of susceptibility to infection by
15. Why are SNPs superior to STR and RFLP for particular phages.
mapping and association studies?
Answer: SNPs are more numerous in the 3. For which of the following organisms must caution
genome than STRs and RFLPs and, therefore, be exercised when evaluating positive PCR results
offer higher resolution for mapping of precise because the organism can be found as normal flora
genome locations. in some patient populations?
a. Neisseria gonorrhoeae
Chapter 11 Study Questions b. HIV
c. Chlamydophila pneumoniae
1. Which of the following genes would be analyzed
d. Streptococcus pneumoniae
to determine whether an isolate of Staphylococcus
aureus is resistant to oxacillin? Answer: Although PCR is specific for S.
pneumoniae, the clinical significance of a
a. mecA
positive result is not certain (d). A significant
b. gyrA
proportion of the population (especially
c. inhA
children) is colonized with the organism, and
d. vanA
PCR cannot discern between colonization and
Answer: S. aureus developed resistance to infection. Neisseria, HIV, and Chlamydophila
antibiotics that target its penicillin-binding are rarely found in healthy populations in the
protein (PBP1) by replacing PBP1 with PBP2a absence of risk factors.
encoded by the mecA gene (a). PBP2a found
in methicillin-resistant S. aureus (MRSA) has 4. Which of the following controls are critical for
a low binding affinity for methicillin. gyrA ensuring that amplification is occurring in a patient
negatively supercoils DNA, favoring replication, sample and that the lack of PCR product is not due
transcription, recombination, and repair. inhA to the presence of polymerase inhibitors?
is an essential enzyme of the mycolic acid
a. Reagent blank
biosynthetic pathway in M. tuberculosis. The
b. Sensitivity control
vanA operon codes for enzymes that modify the
c. Negative control
vancomycin-binding site in vancomycin-resistant
d. Amplification control
enterococci (VRE). Transfer of the transposon
containing the operon to S. aureus produces Answer: The amplification control (d) should
vancomycin-resistant S. aureus (VRSA). always generate a product, even in the
absence of target. The reagent blank monitors results may stay positive long after successful
for contamination. The sensitivity control treatment (a). Advantages of molecular testing
demonstrates that the test is operating properly are rapid methods and minimal sample
at the lower detection levels. The negative requirements.
control ensures the specificity of detection of the
desired target. 7. Which molecular-based typing method has high
typing capacity, reproducibility and discriminatory
5. A PCR assay was performed to detect Bordetella power, moderate ease of performance, and good-to-
pertussis on sputum obtained from a 14-year-old moderate ease of interpretation?
girl who has had a chronic cough. The results
revealed two bands, one consistent with the a. Repetitive elements
internal control and the other consistent with the b. PFGE
size expected for amplification of the B. pertussis c. Plasmid analysis
target. How should these results be interpreted? d. PCR-RFLP
a. These are false-positive results for B. pertussis. Answer: As shown in Table 11.14, PFGE (b)
b. The girl has clinically significant B. pertussis has the performance characteristics listed.
infection. The performance characteristics of repetitive
c. B. pertussis detection is more likely due to elements, plasmid analysis, and PCR-RFLP
colonization. are moderate to good; however, PCR-RFLP
d. The results are invalid because two bands were and repetitive elements are easier to interpret
present. compared with PFGE.
Answer: The girl has clinically significant

B. pertussis infection (b). Molecular analysis 8. A patient has antibodies against HCV and a viral
is used almost exclusively for microorganisms load of 100,000 copies/mL. What is the next test
such as N. gonorrhoea, C. trachomatis, and that should be performed on this patient’s isolate?
B. pertussis. Because the patient shows a. Ribotyping
symptoms of infection and the target-specific b. PCR-RFLP
band is of the correct size, this result is c. Hybrid capture
unlikely to be a false positive if controls
are suitable. Two bands are expected in Answer: Hybrid capture (c) can detect a
the positive case, whereas one band minimum of 4,000 to 5,000 viral genomes.
(amplification control) would be present The test also has the capacity for subtyping.
for a negative result. Ribotyping and PCR-RFLP might be used for
epidemiological studies.
6. Which of the following is an advantage of
molecular-based testing? 9. A positive result for HPV type 16 indicates
a. Results stay positive long after successful a. high risk for antibiotic resistance.
treatment. b. low risk for cervical cancer.
b. Results are available within hours. c. high risk for cervical cancer.
c. Only viable cells yield positive results.
Answer: HPV types 16, 18, 31, 33, 35, 39,
d. Several milliliters of specimen must be
45, 51, 56, 58, 59, 68, 73, and 82 have been
submitted for analysis.
classified as oncogenic and are found to cause
Answer: Molecular testing can be highly anogenital cancers (c). These types are not
sensitive for target nucleic acids, even from associated with specific responses to antibiotic
dead or residual benign organisms, so that test treatment.
10. Which of the following is used to type molds? electrophoresis, and mitochondrial point
mutations (c) can be detected by PCR-RFLP
a. Sequence-specific PCR
or sequencing, large mitochondrial deletions
b. Microarray
are detected with blot hybridization
c. ITS sequencing
techniques.
d. Flow cytometry
Answer: Molds are typed by PCR and 4. A patient was tested for Huntington disease. PCR
sequencing of internal transcribed spacer (ITS) followed by PAGE revealed 25 CAG units. How
regions (c) in 28s RNA. The high resolution of should the results be interpreted?
SSP-PCR and array technology is not indicated
for identifying molds. Microscopic examination a. This patient has Huntington disease.
of morphology provides more information than b. This patient has a 1/25 chance of contracting
would detection by flow cytometry, which would Huntington disease.
require a staining antibody for molds. c. This patient is normal at the Huntington
locus.
d. The test is inconclusive.
Answer: This patient is normal at the HTT
1. Which of the following is not a triplet-repeat locus (c). The frequency of the disorder is 3
expansion disorder? to 7 per 100,000 people of European ancestry
and less for Asian and African ancestries. In
a. Fragile X syndrome Huntington disease, the CAG repeat expands
b. Huntington disease from 9 to 37 repeats to 38 to 86 repeats.
c. Factor V Leiden
d. Congenital central hypoventilation syndrome 5. Which of the following methods can detect the
Answer: Factor V Leiden (c) is a point factor V Leiden mutation?
mutation (1691A→G, R506Q) in the F5 gene a. PCR-RFLP
and not a disorder. Fragile X syndrome results b. SSP-PCR
from expansion of GCC repeats 5′ to the FMR1 c. Invader technology
gene. Expansion of a CAG repeat within the d. All of the above
HTT gene causes Huntington disease. CCHS
is associated with a polyalanine (GCN) repeat Answer: All of the listed methods (d) are
expansion in the PHOX2B gene. capable of detecting a single-nucleotide
change in DNA. PCR-RFLP relies on a
2. A gene was mapped to region 3, band 1, subband restriction enzyme recognition site containing
1, of the long arm of chromosome 2. How would the target nucleotide. In SSP-PCR, the 3′
you express this location from an idiogram? end of one primer hybridizes to the target
nucleotide. Invader technology relies on a probe
Answer: The designation of this location is designed to hybridize to the normal or mutant
2q31.1. sequence.
3. Which of the following can be detected by PCR?

6. The most frequently occurring mutation in the HFE
a. Large mitochondrial deletions gene results in the replacement of cysteine (C) with
b. Full fragile X disorder tyrosine (Y) at position 282. How is this expressed
c. Mitochondrial point mutations according to the recommended nomenclature?
Answer: Whereas full fragile X disorder Answer: The recommended nomenclature is
(b) can be detected with PCR and capillary C282Y.
7. MELAS is a disease condition that results from b. To which allele of D16S539 is the gene
an A to G mutation at position 3243 of the linked?
mitochondrial genome. This change creates a single
Answer: The gene is linked to allele 6 of
ApaI restriction site in a PCR product, including
D16S539.
the mutation site. What would you expect from a
PCR-RFLP analysis for this mutation in a patient How might one perform a DNA analysis for the
with MELAS? presence of the disorder?
a. A single PCR product resistant to digestion with a. Analyze D16S539 for the 6 allele by PCR.
ApaI b. Sequence the entire region of the chromosome
b. A single PCR product that cuts into two where D16S539 was located.
fragments upon digestion with ApaI c. Test as many STRs as possible by PCR.
c. A single PCR product only if the mutation is d. Use a variant-specific test to detect the
present unknown gene mutation.
d. Two PCR products Answer: Because the 6 allele of D16S539
Answer: Amplification of the regions containing is linked to the disease gene, analysis of the
the restriction site will yield one amplicon, D16S539 locus for the 6 allele (a) would be
which will cut into two ApaI fragments in the informative. Sequencing the chromosome
presence of the mutation (b). In the absence of around D16S539 and testing many STR are less
the mutation (and the disease), the PCR product practical ways to find or detect the disease gene
would be resistant to digestion with ApaI. variant. A variant-specific technology would
require identification of the variant allele, which
8. A father affected with a single-gene disorder and is unknown in this case.
an unaffected mother have four children (three 9. Exon 4 of the HFE gene from a patient suspected
boys and a girl), two of whom (one boy and the of having hereditary hemochromatosis was
girl) are affected. Draw the pedigree diagram for amplified by PCR. The G to A mutation, frequently
this family. found in hemochromatosis, creates an Rsa1 site in
exon 4. When the PCR products are digested with
Rsa1, which of the following results would you
expect to see if the patient has the mutation?
a. None of the PCR products will be cut by Rsa1.
b. There will be no PCR product amplified from
the patient DNA.
D16S539, an STR, was analyzed in the family. The c. The patient's PCR product will yield extra
result showed that the father had the 6,8 alleles, bands upon Rsa1 digestion.
and the mother had the 5,7 alleles. The affected d. The normal control PCR products will yield
children had the 5,6 and 6,7 alleles, and the extra Rsa1 bands compared with the patient
unaffected children had the 5,8 and 7,8 alleles. sample.
a. If D16S539 is located on chromosome 16, Answer: The patient's DNA should be
where is the gene for this disorder likely to be amplifiable, and the amplicon will yield extra
located? bands upon Rsa1 digestion (c). Normal DNA
should not be digested at the mutated site.
Answer: Because all affected individuals have
the same D16S539 allele in this limited example, 10. Most people with the C282Y or H63D HFE gene
the gene for this disorder is linked to D16S539 mutations develop hemochromatosis symptoms.
on chromosome 16. This is a result of
a. iron loss. Chapter 13 Study Questions

b. excessive drinking.
c. high penetrance. 1. What are the two important checkpoints in the cell
d. healthy lifestyle. division cycle that are crossed when the regulation
e. glycogen accumulation. of the cell division cycle is affected?
Answer: Genetically, if a disease phenotype Answer: The transition from unreplicated
is frequently present with the DNA variant, DNA into DNA synthesis (G1 to S) and from
then the variant is said to have a high replicated DNA to cell division (G2 to M) are
penetrance (c). Other conditions, such as regulated checkpoints of the cell division cycle.
iron loss or glycogen accumulation, are
phenotypes that may or may not accompany 2. An EWS-FLI-1 mutation was detected in a solid
the hemochromatosis (too much iron). tumor by RT-PCR. Which of the following does
Lifestyle and alcohol intake are behaviors this result support?
that might affect susceptibility to diseases
in general. a. Normal tissue
b. Ewing sarcoma
c. Inherited breast cancer
11. The majority of disease-associated mutations in the
d. Microsatellite instability
human population are
Answer: The EWS-FLI-1 (t;11;22) translocation
a. autosomal dominant.
is found in Ewing sarcoma (b). A translocation
b. autosomal recessive.
would not likely be inherited. Microsatellite
c. X-linked.
instability results from loss of DNA mismatch
d. found on the Y chromosome.
repair functions. Driver mutations such as this
Answer: Most individuals carry recessive translocation would transform normal cells and
disease mutations (b) that may result in thus would not be found in normal tissue.
affected offspring if two disease alleles
are inherited (homozygosity). Dominant 3. Mutation detection, even by sequencing, is not
and sex-linked mutations (which can be definitive with a negative result. Why?
hemizygous in males) may result in
Answer: Mutations may be present outside of
disease states that negatively affect the
the area analyzed. In addition, epigenetic, post-
reproductive health of a population and
transcriptional, and post-translational events
survival; thus, they are selected against
may affect gene function without changing the
over time.
DNA sequence. Furthermore, abnormalities
(gene mutations, epigenetic changes in
12. Bead array technology is most appropriate for expression) in factors that interact with the
which of the following? target gene product may affect its function in
a. Cystic fibrosis mutation detection the absence of mutations in the target gene.
b. Chromosomal translocation detection
c. STR linkage analysis 4. A PCR test for the BCL-2 translocation is performed
d. Restriction fragment length polymorphisms on a patient with suspected follicular lymphoma.
The results show a bright band at about 300 bp for
Answer: Bead arrays are used to detect
this patient. How would you interpret these results?
multiple point mutations such as those found in
cystic fibrosis (a). Translocations, STR analysis, Answer: A PCR method utilizing one primer
and RFLP require size analysis, which is not a on chromosome 18 and one primer on
capability of bead arrays. chromosome 14 would only yield a product
if the t(14;18) translocation (found in follicular a. No bands

lymphoma) is present. The presence of the b. Germline bands plus rearranged bands
product, therefore, supports the diagnosis of c. Smears
follicular lymphoma. The size of the product, d. Germline bands only
300 bp, is within an expected amplicon size
Answer: Rearranged bands represent the gene
range, which depends on the primer-binding
rearrangement in the monoclonal population of
sites and the translocation breakpoint on
tumor cells (b). There should be germline bands
chromosome 18.
present unless all the cells in the sample are
tumor cells. Smears or no bands on Southern
5. Which of the following misinterpretations would
blot are technical difficulties and are not
result from PCR contamination?
interpretable.
a. False positive for the t(15;17) translocation
b. False negative for the t(15;17) translocation 8. Cyclin D1 promotes passage of cells through the
c. False negative for a gene rearrangement G1-to-S checkpoint. What test detects translocation
Answer: Because PCR contamination is the of this gene to chromosome 14?
presence of product, contamination would a. t(14;18) translocation analysis (BCL2/IGH)
result in a false-positive interpretation (a). A b. t(15;17) translocation analysis (PML/RARA)
negative result for a translocation detected by c. t(11;14) translocation analysis (BCL1/IGH)
PCR is the absence of a target amplicon. A d. t(8;14) translocation analysis (MYC/IGH)
negative gene rearrangement analysis results
in a polyclonal pattern (series of bands or Answer: Cyclin D1 is encoded by the BCL1
peaks), which usually does not occur from a gene located on chromosome 11. The test,
contaminating product. therefore, would have to include analysis of
chromosome 11. Translocation to chromosome
6. After amplification of the t(12;21) breakpoint 14 would be detected as t(11;14) (c).
by qRT-PCR, what might be the explanation for
each of the following observations? (Assume that 9. Why is the Southern blot procedure superior to
positive and amplification controls and a reagent the PCR procedure for detecting clonality in some
blank control are included in the run.) cases?
Answer: a. Southern blot requires less sample DNA than
does PCR.
a. All Ct values are reported as undetectable.
b. The PCR procedure cannot detect certain
There is a problem with the amplification,
gene rearrangements that are detectable by
most likely omission of the detection dye or
Southern blot.
probe difficulties.
c. Southern blot results are easier to interpret than
b. Sample and control Ct values are very high.
PCR results.
There is a problem with the sample PCR,
d. PCR results are not accepted by the College of
such as concentration of a critical component
American Pathologists.
required for the PCR reaction.
c. There are Ct values detected in the reagent Answer: The Southern blot can detect all
blanks. The presence of a product in the gene rearrangements, regardless of the
reagent blank, which should have no sequence structure in the immunoglobulin
template, indicates contamination. gene regions (b). Detection of rearranged
genes by PCR requires that the PCR binding
7. What is observed on a Southern blot for gene sites are present in the genes involved,
rearrangement in the case of a positive result? which may be lost due to the rearrangement
process and somatic hypermutation. PCR Answer: KRAS and BRAF gene products are
is technically less difficult to perform and on the same signal transduction pathway. Once
requires less sample than Southern blot. that pathway is activated through mutations
Interpretation of PCR results is comparable to in either gene, there is no selective advantage
interpretation of Southern blot results. Both to generate further activating mutations in the
methods are reviewable by the CAP if properly same pathway.
validated.
14. What enzyme is responsible for continued
10. Interpret the following results from a translocation sequence changes in the immunoglobulin
assay. heavy-chain gene variable region after gene
Are the samples positive, negative, or rearrangement has occurred?
indeterminate? Answer: Activation-induced cytidine deaminase
Answer: alters C residues so that they mispair, forming
Sample 1: This is a negative result. heteroduplexes. The mispaired bases are
Sample 2: This result is indeterminate because resolved by error-prone repair, which may not
the amplification control product is not present. insert the correct bases.
The PCR reaction may not have worked.
Sample 3: This is a positive result. 15. Why are translocation-based PCR tests more
sensitive than IgH, IgL, or T-cell receptor gene-
11. Which of the following predicts the efficacy of rearrangement tests?
EGFR tyrosine-kinase inhibitors? Answer: Gene rearrangements are not tumor
a. Overexpression of EGFR protein specific, so there will be a background of
b. EGFR-activating mutations normal cell gene rearrangements detected by
c. Patient gender the tests performed with consensus primers.
d. Stage of disease Translocations are tumor specific and not found
in normal cells. The absence of background
Answer: Evidence suggests that, unlike, for allows for more sensitive detection of tumor
example, the HER2 protein, overexpression of cells. Gene rearrangements may be detected
the EGFR protein does not predict the efficacy with increased sensitivity using patient-specific
of EGFR TKI. TKI molecules bind better to primers designed to recognize only the tumor
the protein product of the EGFR gene with gene rearrangement in a given patient, but
activating mutations (b) and are more effective even this approach is affected by the ongoing
in their presence. The efficacy of TKI is not evolution of tumor cell populations.
related to gender or stage of disease.
12. What is the advantage of macrodissection in
testing for tumor-specific molecular markers from
1. Which of the following is a high-resolution HLA
paraffin-embedded formalin-fixed tissue sections?
typing result?
Answer: Macrodissection enriches the
a. B27
representation of tumor cells in the test
b. A*02:02–02:09
sample. This increases the sensitivity
c. A*02:12
of the test because normal cells may
d. A*26:01/A*26:05/A*26:01/A*26:15
dilute the tumor-specific markers with
unaffected DNA. Answer: A*02:12 (c) is a high-resolution
typing result indicating the 12th specific
13. Why are KRAS- and BRAF-activating mutations allele of the HLA-A *25 family of alleles.
almost always exclusive of one another? B27 is a low-resolution serological typing
result. A*02:02–02:09 and A*26:01/A*26:05/ typing. HLA-A2 is not an allele designation. The
A*26:01/A*26:15 are medium-resolution N suffix indicates no expression of the protein.
results.
5. A candidate for kidney transplant has a PRA of
2. Which of the following is a likely haplotype from 75%. How will this affect eligibility for immediate
parents with A25,Cw10,B27/A23,Cw5,B27 and transplant?
A17,Cw4,B10/A9,Cw7,B12 haplotypes?
Answer: A transplant candidate with a %PRA
a. A25,Cw10,B27 activity of more than 50% is considered to be
b. A25,Cw5,B27 highly sensitized. Finding a crossmatch-negative
c. A23,Cw4,B12 donor will be difficult in this case.
d. A17,Cw4,B27
Answer: Because alleles in a haplotype do 6. An SSOP probe recognizes HLA-DRB*
not separate and recombine, only haplotype 03:01–03:04. Another probe recognizes
A25,Cw10,B27 (a) will be inherited from the HLA-DRB*03:01/03:04, and a third probe
parents with the listed haplotypes. A25,Cw5,B27 hybridizes to HLA-DRB*03:01–03:03. Test
would require two recombination events specimen DNA hybridizes to all except the third
between A25,Cw10,B27 and A23,Cw5,B27. probe in a reverse dot-blot format. What is the
A17,Cw4,B27 would require a single exchange HLA-DRB type of the specimen?
between A17,Cw4,B10 and A23,Cw5,B27 after Answer: The test specimen does not hybridize
fertilization. A23,Cw4,B12 would require to HLA-DRB*03:01–03:03, ruling out those
exchanges between three of the haplotypes. types. The other two probes both recognize
HLA-DRB*03:04, which is the HLA-DRB type
3. Upon microscopic examination, over 90% of cells based on these observations.
are translucent after a CDC assay. How are these
results scored according to the ASHI rules? 7. What is the relationship between alleles
Answer: This observation is interpreted as HLA-A*10 and HLA-A*26(10)?
negative for the presence of the test antibody, Answer: HLA-A*10 is the parent allele of
with a score of 1. Less than 10% of the cells HLA-A*26(10). Number designations of new
have taken up the dye. Cytotoxicity (dye uptake) alleles of a previously defined broad specificity
will only occur in those cells that carry antigens or parent allele follow the parent allele in
to the test antibody. parentheses.
4. An HLA-A allele is a CTC to CTT (leu → leu) change
8. A CDC assay yields an 8 score for sera with the
at the DNA level. How is this allele written?
following specificities: A2, A28 and A2, A28, B7,
a. HLA-A*02 and a 1 score for serum with an A2 specificity.
b. HLA-A*02:01:01 What is the HLA-A type?
c. HLA-A2
Answer: This is type A28. The high toxicity
d. HLA-A*02N
reading (>6) in the wells containing A2,
Answer: The first field of digits after the gene A28 sera and A2, A28, B7 sera suggest that
name is the serological type. The next field the cells being tested have surface antigens
(after the colon) is the subtype, where nucleotide matching the A28 antibodies.
substitutions that change the amino acid
sequence are indicated. A third field of digits, as 9. HLA-DRB1*15:01 differs from DRB1*01:01 by a
in HLA-A*02:01:01 (b), designates changes in G to C base change. If the sequence surrounding
the DNA sequence that do not change the amino the base change is … GGGTGCGGTTGCTGG
acid sequence. HLA-A*02 is a low-resolution AAAGAT … (DRB1*01:01) or … GGGTGCGG
TTCCTGGAAAGAT … (DRB1*15:01), which of Answer: The results from the PCR cannot
the following would be the 3′ end of a sequence- be accepted. Check the original nucleic
specific primer for detection of DRB1*15:01? acid preparation by fluorometry or gel
electrophoresis. If it is adequate, repeat the
a. … ATCTTTCCAGGAACCC
amplification. If not, re-isolate the nucleic acid.
b. … ATCTTTCCAGCAACCC
c. … ATCTTTCCAGC
d. … ATCTTTCCAGG 3. An isolated DNA sample is to be stored for at least
6 months.
Answer: SSP-PCR requires complementarity
between the 3′ end of the primer and the Answer: Store the DNA at –70°C in a tightly
template. Primer … ATCTTTCCAGG (d) would sealed tube.
detect … GGGTGCGGTTCCTGGAAAGAT
… (DRB1*15:01). Primer ATCTTTCCAGC is 4. A bone marrow specimen arrives at the end of
identical but not complementary to the template a shift and will not be processed for the Bcl2
sequence. Primers ATCTTTCCAGGAACCC translocation until the next day.
and ATCTTTCCAGCAACCC do not end
on the polymorphic base that defines Answer: Hold the specimen at refrigeration
DRB1*15:01. temperature.
10. The results of an SSP-PCR reaction are the 5. The temperature of a refrigerator set at 8°C (±2°C)
following: lane 1, one band; lane 2, two bands; lane reads 14°C.
3, no bands. If the test includes an amplification
Answer: Recheck the temperature after a few
control multiplexed with the allele-specific primers,
hours while considering alternate refrigeration
what is the interpretation for each lane?
locations. If the temperature does not return
Answer: Lane 1 is a negative result because the within range, notify the supervisor and
one band is the amplification control. Lane 2 is relocate sensitive materials to the alternate
a positive result for the allele detected in that location.
reaction. Lane 3 is a failed PCR and therefore
not informative. 6. A PCR test for the BCR/ABL translocation was
negative for the patient sample and for the
sensitivity control.
Answer: Repeat the PCR with the addition of a
What actions should be taken in the following situations? new sensitivity control.
1. An unlabeled collection tube with a requisition 7. A fragile X test result has been properly reviewed
for a factor V Leiden test is received in the and reported.
laboratory.
Answer: Securely file the test results,
Answer: Notify the laboratory supervisor and documents, and associated images electronically
reject the specimen. All primary specimen or as hard copies in the laboratory archives.
containers, regardless of accompanying
documents, require a label carrying at least two
8. A bottle of reagent alcohol with a 3 in the red
patient-specific identifiers.
diamond on its label is to be stored.
2. After PCR, the amplification control has failed to Answer: Place the bottle of alcohol in a safety
yield a product. storage cabinet for flammable liquids.
9. The expiration date on a reagent has passed. 10. Test results are to be faxed to the ordering
physician.
Answer: Discard the reagent. If the material
can be used for nonclinical purposes, such as Answer: Fax the results with a cover sheet
training exercises, label and store the reagent containing the proper disclaimers.
in a separate area away from patient testing
reagents.
Appendix B
Answers to Case Studies
introduced into one of the patient’s sections during

Chapter 10 tissue processing.
Case Study 10.1 Interpretation
The woman was successfully engrafted. The recipient Chapter 11
peak pattern has converted mostly to the donor peak Case Study 11.1 Interpretation
pattern at 100 days. The percentage of residual recipient
cells can be calculated by the analysis of unshared infor- The molecular evidence is consistent with norovirus.
mative alleles at this locus: Norovirus cannot be cultured. Laboratory tests include
electron microscopy, serology, and RT-PCR. Electron
R unshared
%R = × 100 microscopy and immune electron microscopy require
( R unshared + D unshared ) highly specialized equipment and advanced technical
=
3,171
× 100 = 7.2% expertise. Interpretation of tests for serum antibod-
(3,171 + 40, 704) ies to the virus is not always straightforward because
most of the adult population has serum antibodies to
The patient was (100 − 7.2) = 92.8% donor at 100 days,
this virus. Furthermore, the development of detectable
and at 1 year, no residual recipient alleles are present at
IgM antibodies may take several days after exposure.
the level of detection of the instrument (0.5% to 1.0%).
RT-PCR, therefore, is the method of choice for detec-
At this time, the patient is reported to have more than
tion of this RNA virus. The test targets the viral RNA
99% donor cells and less than 1% recipient cells in
polymerase gene, which can be used for a broad spec-
the test specimen. The patient is therefore successfully
trum of noroviral types. The results in this case showed
engrafted with donor cells.
that the virus was present in the salad lettuce served at
the hotel. Lettuce sampled directly from the distributor
did not carry the virus, indicating that the contamination
These results indicate that the two brothers are identi- occurred at the hotel. This observation was consistent
cal twins. This means that STR analysis cannot be used with the discovery of the virus in the hotel workers who
for monitoring engraftment after transplant in this case. prepared the salad. Direct sequencing of cDNA prepared
Advances in epigenetics may lead to discerning molec- from viral RNA revealed identical sequences for all pos-
ular characteristics even in identical twins, but not in itive specimens, showing that the guests, workers, and
current practice. With regard to prognosis, there is less food source shared the same viral strain.
chance of graft-versus-host disease in this case, but there
is also no graft-versus-tumor effect that can improve the Case Study 11.2 Interpretation
chance of removing all of the recipient tumor cells.
Results from culturing the lysates were consistent with
multidrug resistant Staph aureus (MRSA). The pulsed
field gel electrophoresis (PFGE) analysis revealed that
The results show that the material is not of the same all except one of the isolates from the students were
genetic origin as that of the patient. Apparently, from the same strain. One isolate exhibited two PFGE
this microscopic piece of tissue was a contaminant differences from the others, indicating that it was closely
501
502 Appendix B • Answers to Case Studies
related to these S. aureus isolates. Resistance to oxacillin/ premutation is not detectable by Southern blot. Capil-
methicillin results from the expression of an altered lary electrophoresis with triplet-primed PCR produces
penicillin-binding protein encoded by the mecA gene. an extended peak pattern for the premutation. See Figure
All five isolates shared the type IV mecA gene asso- 12.24B.
ciated with MRSA. The toxin encoded by Panton-
Valentine Leukocidin (gene) (PVL), also produced by Case Study 12.2 Interpretation
these isolates, is thought to be responsible for tissue
necrosis in MRSA infections. The 16,000-bp product is the linearized normal mito-
Further investigation into the cases revealed that all chondrial DNA (16,500 bp). The 11,500-bp product is the
of the affected students had participated in a wrestling linearized mitochondrial circle with a 5,000-bp deletion.
meet at one of the high schools. Passage of the organ- The deletion is associated with Kearns–Sayre syndrome
ism during this event was the likely source of the shared (KSS), a rare neurological condition affecting muscle
infections. The meet location was thoroughly cleaned function, stature, and hearing, among other symptoms.
according to the Centers for Disease Control and Pre- The results shown are a case of heteroplasmy because
vention recommendations, and the students were encour- both normal and deleted mitochondria are present. The
aged to practice diligent hand washing and optimal number of deleted mitochondria compared with the
hygiene. number of normal mitochondria will affect the severity
of the disease symptoms.
The observation that the patient’s viral loads were grad-
ually rising was a sign that the virus was developing The PCR-restriction fragment length polymorphism
resistance to the antiviral therapy. Variations of viral (RFLP) results in lane 5 indicate that the patient has
quantity within 0.3 log units are considered normal. the HFE C282Y mutation, the most common inherited
This patient, however, was seeing significant increases mutation hemochromatosis. The G → A base change
in viral replication over the last 6 months. produces an additional recognition site for the RsaI
Genotyping showed that this patient’s virus has a restriction enzyme, 30 bp from the end of the 140-bp
mutation in the reverse transcriptase gene that has ren- amplicon in the normal sequence. The absence of the
dered the virus resistant to AZT. The patient’s treatment 140-bp fragment in lane 5 shows that the mutation is
should be changed, replacing AZT with another reverse homozygous (although hemizygosity will also yield this
transcriptase inhibitor that would be unaffected by the pattern). The symptoms are likely due to iron overload
mutation, such as didanosine or lamivudine. Viral load caused by the loss of regulation of iron load through the
measurements will be taken regularly to ensure that the HFE protein.
change in therapy causes a decrease in viral load over
the next few months. Further genotyping may be per- Case Study 12.4 Interpretation
formed if the viral load starts to trend up again.
The results of the physical and chemistry tests indicate
that this patient has a thrombophilic clotting disorder
Chapter 12 (hypercoagulation). The molecular tests show a homozy-
gous (or hemizygous) factor V Leiden F5 1691 G → A
mutation. This nucleotide substitution in conjunction
The results in lane 3 show an expansion of the FMR1 with the special primers used for this test will produce
gene promoter, suggesting a premutation in the mother. a HindIII site in the F5 exon 10 PCR product. Results
The full fragile X mutation can be detected by poly- for the prothrombin 20210 G → A mutation are nega-
merase chain reaction (PCR) and capillary electropho- tive (homozygous normal). The mutation in the Factor
resis. The cytogenetic results indicate the presence of 5 protein interferes with its ability to block the clotting
the fragile X expansion. The size shift caused by the process, resulting in the symptom of thrombophilia.
Appendix B • Answers to Case Studies 503
Chapter 13 changes do not change the amino acid sequence, and

some sequence changes are benign to protein function,
Case Study 13.1 Interpretation so the sequencing information is required for defini-
The molecular results revealed a small population of tive interpretation of the pathogenicity of the sequence
cells in the bone marrow specimen with the t(14;18) change.
translocation. It was apparently below the level of detec-
tion of the flow-cytometry and cytogenetics tests for this Case Study 13.4 Interpretation
specimen with minimal B cells. The PCR translocation The microsatellite instability (MSI) analysis shows
analysis, however, is lacking amplification controls. instability in at least two of the five microsatellites
There may be a small monoclonal population repre- tested (BAT25 and BAT26). The peak pattern (additional
senting part of the lymphoid aggregates observed in the alleles) in the tumor cells compared with normal cells
morphological studies. The complete blood count (CBC) from the same patient results from loss of nucleotides
is not representative of this cell population. The immu- in the repeated sequences. Normally, the losses would
noglobulin gene-rearrangement results indicate that the be repaired by the mismatch repair system, a protein
gene rearrangement in this small abnormal cell popula- complex that includes MLH1, MSH2, MSH6, PMS2,
tion is not amplifiable or is below the level of detection and other proteins. If the repair system malfunctions
of this assay. Although detection of the t(14;18) translo- from the loss of one or more of its component parts, the
cation is consistent with follicular lymphoma, the clin- additional alleles will be replicated in ensuing rounds of
ical significance of this cell population is not apparent replication. According to the National Cancer Institute,
from a single encounter. Patient history and further anal- high MSI is defined by instability in at least two of five
yses will provide information as to whether these results NCI-designated microsatellite loci. If more than five
are arising from molecular remission or early signs of microsatellite loci are used to test for MSI, a cut point
relapse. of 30% of the markers tested is considered MSI. New
methods to detect MSI in DNA utilize qPCR or NGS
Case Study 13.2 Interpretation detection of insertions or deletions in other microsatel-
The band pattern in the normal control lane represents lite sequences.
the products of the outer primers and one inner primer
complementary to the normal JAK2 sequence. The Case Study 13.5 Interpretation
pattern in lane 3 shows an additional product primed The t(9;22) translocation is present in 95% of cases of
by the JAK2 V617F mutation–specific primer. The chronic myelogenous leukemia (CML), 25% to 30%
results in lane 2 indicate that the patient does not have of adult acute lymphoblastic leukemia (ALL), and 2%
the V617F mutation. This test only detects the targeted of pediatric ALL. The presence of the translocation is
V617F mutation. It does not rule out the presence of a negative prognostic factor in adult ALL. The t(1;19)
other mutations in JAK2 exons 12 to 14, which could be translocation may be present in pre-B-ALL. Monoclonal
assessed as a reflex test, if indicated. immunoglobulin heavy-chain and light-chain gene rear-
rangements may be used to monitor B-ALL. Monoclonal
Case Study 13.3 Interpretation T-cell receptor gene rearrangements monitor for T-ALL.
The single-strand conformation polymorphism (SSCP)
band pattern in lane 2 is different from that of the normal
control in lane 3, indicating the presence of a sequence Chapter 14
alteration in the patient DNA. The additional band rep-
resents a conformer formed by the folding of the altered
sequence. SSCP shows the presence and general loca- This patient has DQ alleles, DQA1*05:01 and
tion of a sequence variant. Direct sequencing identifies DQB1*02:01, that form the two parts (alpha chain and
the particular nucleotide change. Synonymous DNA beta chain, respectively) of the DQ2 variant heterodimer
504 Appendix B • Answers to Case Studies
often found in people with celiac disease. Although a and a single mismatch is less associated with graft-
diagnosis cannot be made based on the HLA haplotype versus-host disease (GVHD) than multiple mismatches
alone, the presence of the variant allele is consistent in the class I and II antigen loci. The actual effect of
with such a diagnosis made based on clinical and moran allele on the immune response will also depend
phological evidence. on the location of the polymorphic amino acid in the
protein. A mismatch may be tolerated if the polymorphic
Case Study 14.2 Interpretation amino acid is not exposed on the outer surface of the
folded protein.
A successful transplant does not absolutely require a
fully matched donor. In some cases, there may be more
risk in delaying a transplant in search of a fully matched
donor than in proceeding with the transplant earlier in the A parent’s alleles will not be the same as the offspring’s.
disease process. Mismatches at the HLA-B and HLB-C Even before typing, the mother will be expected to share
loci may be better tolerated than those at the HLA-A at least half of the daughter ’s alleles. A fully matched
and HLA-DRB1 loci. Donor 3 is the closest match at the sibling is the best donor type. Due to the undefined
HLA-A, HLA-B, and HLA-DRB1 loci. Because donor 3 factors that can affect organ rejection, fully matched
is homozygous for the A locus, only one determinate is siblings are also better than unrelated fully matched
displayed to the recipient immune system, which could donors for successful transplant. In the absence of a
lessen the likelihood of graft rejection. Conversely, when fully matched donor, the mother is an acceptable donor
the graft does not share one of the recipient alleles, donor for her daughter. Crossmatching results from the daugh-
T cells may react against the recipient, and graft-versus- ter ’s serum were negative for antibodies against antigens
host disease may occur. In the present case, however, displayed on the mother ’s donated organ, favoring suc-
there is only one mismatch among the tested alleles, cessful transplantation.
Glossary
A alkaline phosphatase an enzyme frequently used

to generate signals from chemiluminescent or
A site adjacent site; location of incoming charged chromogenic substrates
tRNA binding in the ribosome complex for
allele a different version of the same sequence, gene,
acceptance of the growing peptide
or locus
abasic site a position on the DNA sugar-phosphate
allele dropout missing sequence data due to loss of
backbone where the ribose sugar does not carry a
DNA fragments during library preparation
nitrogen base
allele-specific oligomer hybridization mutation/
absorptivity constant the characteristic tendency of
polymorphism detection technique using
a substance to absorb light at a given wavelength;
immobilized target and sequence-specific probes
used in quantifying nucleic acids, the absorptivity
constants of DNA and RNA are (50 ug/mL)/ allelic discrimination mutation/polymorphism
absorbance unit and (40 ug/mL)/absorbance unit, detection using real-time PCR and fluorogenic
respectively, at 260 nm wavelength probes
acrocentric having the centromere nearer to one end allelic exclusion expression of a gene on only one of
of the chromosome than the other two homologous chromosomes, with the other not
expressed
adenine one of the common nitrogen bases in DNA
and RNA allelic ladder a set of PCR amplicons of a VNTR
or STR that represents all possible alleles in a
adenosine one of the common nucleosides in DNA
population
and RNA
alloantibodies antibodies that recognize human
affinity maturation selection of B cells expressing
antigens
antibodies with greater affinity of the antigen
allogeneic genetically from different individuals
agarose polymer of agarobiose (1,4-linked
3,6-anhydro-α-L-galactopyranose) that, when allograft a transplanted organ from a genetically
hydrated, forms a gel frequently used for sieving different donor
nucleic acids alpha phosphate the phosphate group closest to the
alignment comparison and lining up of two or more ribose sugar in a nucleotide
nucleic acid or protein sequences to achieve the alpha satellite highly repetitive sequence found at the
maximal number of identical positions centromere
505
506 Glossary
alternative splicing removal of introns from RNA analytic sensitivity lower limit of detection of an
using different breakpoints analyte
Alu elements short interspersed nucleotide sequences analytic specificity ability to detect only the analyte
containing recognition sites for the Alu restriction and not nonspecific targets
enzyme anaphase stage in mitosis or meiosis where replicated
ambiguity recognition of two or more antigens by the chromosomes separate
same antibody ancestral haplotype the haplotype in which a
amelogenin locus a gene located on the X and mutation originally occurred
Y chromosome with an XY-specific polymorphism Anderson reference sequence of the mitochondrial
aminoacyl tRNA synthetases enzymes that hypervariable regions used as a reference for
covalently attach the appropriate amino acid to defining polymorphisms
its tRNA aneuploid having an aberrant number of
amino acid tRNA synthetase one of 20 enzymes that chromosomes per nucleus, caused by genome
attach the appropriate amino acid to its anticodon- mutations
containing tRNA aneuploidy in diploid organisms, having other than
amino acids nitrogen-containing molecules with two of each chromosome
specific biochemical properties that are the building anion negatively charged atom or molecule
blocks of protein
annealing hybridization of complementary sequences
amino-terminal the end of a protein or peptide
containing the amino group of the amino acid, annotation classification of sequence variants based
usually considered the beginning of the protein’s on their biological and/or clinical significance
amino acid sequence anode positively charged electrode to which anions
ammonium persulfate APS; with TEMED, catalyzes migrate
the polymerization of acrylamide gels anticipation in genetics, rising phenotypic severity
amplicon the product of a PCR reaction through generations of a family
amplification replication, copying antigen retrieval protease treatment of tissue to
uncover target epitopes
amplification control template sequences that are
always present; used to confirm the functional anti-human antibodies AHA; alloantibodies;
competence of the reaction mix antibodies that recognize human antigens
amplification program conditions under which antimicrobial agent antibiotic; a substance that
a PCR reaction occurs, including denaturation, inhibits growth of or kills bacteria
annealing, and extension temperatures and times antimir a synthetic short RNA that antagonizes
amplified fragment length polymorphism AFLP; oncomirs through complementary hybridization
analysis of polymorphic DNA by amplification of antiparallel two complementary single-stranded
selected regions and resolving the amplified products nucleic acids oriented so that when hydrogen-bonded
by size together through complementary bases, the 5′ end of
analyte measurement range AMR; the range within one molecule is next to the 3′ end of the other
which a specimen may be tested directly (without antisense strand the single strand of a DNA double
dilution or concentration) for an analyte helix that is used as a template for messenger RNA
analyte-specific reagents ASRs; test components that synthesis
detect a specific target apoptosis self-directed cell death
analytic accuracy production of correct (true-positive arbitrarily primed PCR a PCR method for
and true-negative) results amplification of complex targets using random
Glossary 507
priming with 6- to 10-base primers for typing of banding in cytogenetics, the formation of
organisms; also known as randomly amplified microscopically visible bands on chromosomes by
polymorphic DNA (RAPD) staining
arm in cytogenetics, one end of a chromosome, from bar coding attachment of short (6-8 base) sample-
the centromere to the telomere specific DNA sequences to sequencing templates
array in molecular biology, a set of probes or targets allowing multiple samples to be sequenced together;
immobilized on the same substrate also called indexing
autologous genetically from the same individual Barr body a structure visible in the interphase
autophagy degradation of intracellular substrates nucleus formed by the inactive X chromosome in
within the cell female mammals
autoradiograph an image produced by exposing a basal transcription complex a complex of protein
light-sensitive film to a light- or radiation-emitting factors required by RNA polymerase to initiate RNA
membrane synthesis
autosomal dominant an inheritance pattern where base nitrogen base; also used as an expression of
the child of an affected and an unaffected individual units of single-stranded nucleic acid
has a 50% probability of being affected base extension sequence scanning BESS; mutation/
autosomal recessive an inheritance pattern where polymorphism scanning using dUTP incorporation
the child of an affected and an unaffected carrier into DNA followed by enzymatic cleavage at the
has a 50% probability of being affected and where dU sites, followed by electrophoretic resolution of
the child of two affected individuals has a 100% the resulting fragments
probability of being affected base pair an expression of units of double-stranded
autosomal STR short tandem repeats located on nucleic acid
other than the X and Y chromosomes base pairing association of adenine with thymine/
autosome any chromosome except for the X and uracil or guanine with cytosine through specific
Y sex chromosomes hydrogen bonds
avuncular testing determination of the probability basic local alignment search tool BLAST; a
of an aunt/uncle–niece/nephew relationship through sequence-comparison algorithm
genetic polymorphisms
bDNA branched DNA; a signal amplification system
bead array probes attached to fluorescence-labeled
B beads
bacteriocidal kills bacteria beta-2 microglobulin a 12-kD peptide associated
bacteriophage viruses that infect bacteria at the cell membrane with the MHC heavy chain
bacteriostatic prevents growth of bacteria comprising the class I HLA
balanced in cytogenetics, genetic events that result in beta-lactam a cyclic protein found in antibiotics
no gain or loss of genetic material beta-lactamase an enzyme produced by bacteria
balanced polymorphism a DNA sequence difference, that are resistant to β-lactam–containing
the phenotypic effect of which is counteracted by a antibiotics
second trait or polymorphism betaine N,N,N-trimethyl glycine; used in
band in molecular biology, a pattern on a gel or PCR reactions to increase specificity and yield by
autoradiogram representing a fragment of nucleic facilitating strand separation of the target double
acid or protein helix
band compression bunching of fragments in gel bidirectional reading or analysis of both forward and
electrophoresis migration reverse strands of a double-stranded DNA
508 Glossary
bin in fragment analysis, a defined range of error Cambridge reference sequence sequence of the
expected for electrophoretic migration of the same mitochondrial hypervariable regions used as a
DNA fragment reference for defining polymorphisms
binning in fragment analysis, the collection of cancer diseases that are caused by uncoordinated,
all peaks or band migration distances within a rapid cell growth
characteristic distribution in order to determine if cap 7-methyl G 5′ppp 5′ G/A covalently attached to
two peaks or migration distances are representative the 5′ end of messenger RNA
of the same allele
capillary electrophoresis separation of particles in
bioinformatics an adaptation of information solution inside of a glass capillary
technology for the biological sciences;
capillary gel electrophoresis separation of particles
computational biology
through a sieving polymer or gel inside of a glass
biotin a vitamin that is used in the laboratory for
capillary
nonradioactive probe detection
capillary transfer movement of nucleic acid or
bisulfite (DNA) sequencing nucleotide sequence
protein from a gel matrix to a membrane by
analysis of DNA that has been treated with sodium
capillary action
bisulfite; a method to detect methylated cytosines
in DNA carboxy-terminal the end of a protein or peptide
blank a reaction mix containing all components containing the carboxyl group of the amino acid,
except for target; used to detect target contamination usually considered the end of the protein’s amino
in test reagents acid sequence
BLAST basic local alignment search tool; a software carcinoma tumors of epithelial tissue origin
program that compares nucleotide or protein cathode negatively charged electrode to which
sequences to find regions of similarity between them cations migrate
blocking covering nonspecific binding sites before cation positively charged atom or molecule
probe hybridization cell division cycle the succession of events when a
blot transfer; the membrane carrying the transferred single cell divides, including pre-replicative phase
nucleic acid or protein (G1), DNA synthesis (S), post-replication (G2), and
BOX palindromic repetitive elements found in separation (M)
Streptococcus pneumoniae centromere specialized, repetitive DNA sequence
break-apart probe FISH probes designed to detect that attaches to the spindle apparatus through the
translocations where there are multiple possible kinetochore
partners centromeric probe CEN, CEP; a probe that binds to
buffer an ionic substance or solution that maintains centromeres
specific pH chain a term sometimes used to describe a polymer
buffy coat white blood cells separated by density of nucleotides comprising a single strand of nucleic
centrifugation acid
chaperone a specialized protein that protects growing
C peptides as they emerge from the ribosome
C banding centromere staining charging attachment of an amino acid to tRNA
calibration fitting an instrument test output with the checkpoint regulated places in the cell division cycle;
actual amount of a reference analyte between the G1 and S phases (G1 checkpoint) and
calibration verification testing of materials of known between the G2 and M phases (G2 checkpoint)
amounts throughout the measurement range to chemical cleavage breaking DNA chains with
ensure instrument accuracy specific nonenzymatic substances
Glossary 509
chimera an individual harboring cells or tissue from comb in electrophoresis, a device placed in melted
two different zygotes agarose to mold wells or spaces for introducing
chromatin relaxed chromosomes found in interphase samples to the gel
nuclei combined paternity index CPI; the product of the
paternity indexes for a set of alleles
chromosome a DNA double helix that carries genes
combined sibling index likelihood ratio generated
chromosome paint cytogenetic probe analysis
by a determination of whether two people share one
designed to visualize an entire metaphase
or both parents; also called sibling index, kinship
chromosome
index
chromosome spread an array of intact chromosomes compaction wrapping, folding, and coiling DNA to
displayed upon hypotonic lysis of a nucleus fit inside the nucleus
class-switch recombination movement of the VDJ comparative genome hybridization CGH;
gene segment of a rearranged immunoglobulin gene a microarray method to detect deletions or
to gene segments encoding IgG, IgE, or IgA constant amplifications in DNA
regions
comparative genomic array an array hybridized to
cleavage-based amplification a signal-amplification DNA in order to detect insertions or deletions
system based on the proprietary cleavase enzyme complement a group of proteins that, in combination
Clinical Laboratory Improvement Amendments with antibodies, causes the destruction of particulate
CLIA; recommendations passed by Congress in antigens
1988 to establish quality and testing standards complement-dependent cytotoxicity CDC; killing of
clinical sensitivity ability of a test result to predict a cells based on HLA type using reference antigens
clinical condition complementarity-determining region CDR; a
clinical specificity disease-associated results only in domain in the immunoglobulin heavy-chain gene
patients who actually have the disease conditions variable-region coding for amino acids that directly
contact antigen
clone two or more cells or individuals having the
same genotype complementary a term used to describe the order of
two antiparallel single-stranded nucleic acids such
clonotype the nucleotide sequence of a surface- that A on one strand is always across from T on the
expressed T-cell receptor gene rearrangement other strand, and C is always across from G
coa typing tandem repeat element in the 3′ coding complementary DNA cDNA; double-stranded DNA
region of the Staphylococcus aureus coagulase gene, synthesized using RNA as a template
coa; analyzed by PFGE to identify MRSA
computational biology application of statistics,
CODIS Combined DNA Indexing System; a set of mathematics, and computer science to the study of
13 STR loci and amelogenin used for positive living systems
human identification conformation-sensitive gel electrophoresis
codominant offspring simultaneous expression of resolution of homoduplexes from heteroduplexes
both parental phenotypes under specified gel conditions
codon a three-nucleotide sequence that will guide the conformer a three-dimensional structure formed by
insertion of a specific amino acid into protein folding and intrastrand hybridization of a single
coefficient of variance CV; a normalized measure strand of nucleic acid
of dispersion about the mean; an expression of test congenital “born with”; having a genetic component
precision determined through repeated tests of the conjugate a structure comprised of more than
same sample analyte one type of molecule, such as a glycoprotein or
510 Glossary
lipoprotein, or an antibody with covalently attached control analytes of known type and amount that are
alkaline phosphatase included with test specimens to monitor method
conjugated proteins proteins with lipid, systems
carbohydrate, metallic or other components coverage the number of times a variant is sequenced
conjugation transfer of genetic information by in a sequencing reaction
physical association of cells CRISPR clustered regularly interspaced short
palindromic repeat sequences that provide bacterial
consensus primer a primer directed to the
immunity to bacteriophage or exogenous plasmids
immunoglobulin or T-cell receptor genes with
crossmatching analysis of the reaction between
sequences representing the most frequently occurring
recipient serum antibodies and donor lymphocytes
nucleotide at each position
cross-reactive epitope group CREG; human
consensus sequence a family of sequences leukocyte antigens recognized by antibodies that
representing different variations in a population but also recognize other HLAs
with similar motifs in nucleotide order crRNA short, mature RNAs synthesized from
conservation amino acid (or nucleotide) substitutions CRISPR regions (processed from pre-crRNA)
that preserve the biochemical and physical properties carrying complementary sequences to a target DNA
of the original residue or nucleotide sequence cryostat an instrument similar to a microtome that
conservative (1) in DNA replication, a term used to allows cutting slices of frozen tissue at freezer
describe replication of both strands of the parent temperatures
double helix simultaneously, producing a daughter cryotube plastic storage tubes designed for storage at
double helix comprising two replicated strands; freezer temperatures
(2) in protein sequences, substitution of an amino Cot value expression of the complexity of DNA
acid with one of similar biochemical properties sequences
conservative substitution an alteration in the Cot1/2 time required for half of a double-stranded
nucleotide sequence that results in the substitution of sequence to anneal under a given set of conditions
an amino acid with one of similar properties cut-off value a quantitative assay level that
constitutive transcription that is constantly active distinguishes positive from negative results; also
contact precautions protective equipment and/or called cut point
clothing used for direct patient contact and potential cycle in PCR, one series of extension, annealing, and
exposure to airborne or contact-transmissible extension
infectious agents from the patient cycle sequencing semiautomated method to
determine the order of nucleotides in a nucleic acid
contamination presence of unwanted materials; in
using a thermal cycler
PCR, presence of extraneous, nontarget template
cycling probe a signal-amplification system based
DNA in a reaction solution
on RNase-H digestion of probe bound to target
contamination control action taken to avoid or sequences
detect unwanted substrates or products of a reaction; cytidine one of the common nucleosides in DNA and
in PCR, a reaction mix containing all components RNA
except target cytochrome P-450 a group of enzymes in the
continuous allele system genetic structures that vary endoplasmic reticulum that participates in enzymatic
by increments and are defined by these incremental hydroxylation reactions and also transfers electrons
variations to oxygen
contract precautions protective equipment and/ cytopathic effect CPE; lysis, death, or growth
or clothing used with direct patient contact inhibition of cells due to viral infection
and potential exposure to airborne or contact- cytosine one of the common nitrogen bases in DNA
transmissible infectious agents from the patient and RNA
Glossary 511
D discrete allele system a set of alleles that can be

classified by a finite number of types
DAPI 4′,6-diamidino-2-phenyl indole; DNA-specific
dye, used to visualize nuclei discriminatory capacity DC; the power of a locus
for use in identification, considering the number of
de novo sequencing determination of nucleotide
different alleles of the locus and the total number
order for the first time
of individuals tested in a given population
deazaGTP a modified nucleotide used to destabilize
discriminatory power ability of typing methods to
secondary structure in template DNA
clearly distinguish strains
deletion loss of genetic material
dispersive a term used to describe DNA replication
denaturation in nucleic acids, loss of hydrogen where both strands of the parent double helix
bonding between complementary strands; in protein, undergo simultaneous, discontinuous replication
loss of tertiary structure
dNTP deoxyribose nucleotide triphosphate
denatured alcohol ethanol mixed with other
components domain a functional part of a protein
denaturing agent in electrophoresis, formamide dominant Mendelian inheritance pattern where
or urea used to remove secondary structure from offspring of an affected individual and an unaffected
molecules mate have a 50% to 100% risk of expressing the
affected phenotype
denaturing gel a polyacrylamide gel containing a
substance that will separate double-stranded DNA dominant-negative loss of function of multimeric
into single strands proteins by interaction with one abnormal
monomer
deoxyribose ribose without a hydroxyl group on the
carbon in the 2 position on the sugar ring dot blot hybridization technique with multiple targets
spotted on a membrane all exposed to the same
depurination removal of purines from the sugar-
probe
phosphate backbone of DNA
dual-fusion probe dual-color probe; FISH probe
derivative chromosome an abnormal chromosome
designed to detect translocations and their reciprocal
comprising rearranged parts from two or more
products
unidentified chromosomes joined to a normal
chromosome dye blob artifactual peak pattern caused by residual
unincorporated labeled dideoxynucleotides in a
detection limit lower limit of detection of the
analyte sequencing reaction
dideoxy chain termination a method using dye primer covalent attachment of fluorescent
dideoxynucleotides to determine the order or molecules to a sequencing primer
sequence of nucleotides in a nucleic acid dye terminator covalent attachment of fluorescent
dideoxy DNA fingerprinting ddF; mutation/ molecules to dideoxynucleotides
polymorphism screening using one or two
dideoxynucleotides and gel electrophoresis E
dideoxynucleotide ddNTP; a nucleoside triphosphate
E-value in searching for specific sequences, expect
lacking a hydroxyl group at the 3′ ribose position
value (E) describes the number of sequence matches
diethyl pyrocarbonate ribonuclease inactivator; one can “expect” to see by chance when searching a
DEPC cross-links RNase proteins database of a particular size
digoxygenin steroidal compound used for eastern blot a hybridization technique used to detect
nonradioactive probe detection post-translational modification of immobilized
diploid having two of each chromosome proteins
512 Glossary
electrode a metallic conductor of an electrical circuit bacteria; typing of bacteria by analysis of PCR
that includes nonmetallic parts amplicons produced from primers homologous to
electroendosmosis solvent flow toward an electrode these sequences
in opposition to particle migration E site position in the ribosome from which the
electrokinetic injection loading of nucleic acid for “empty” tRNA is released
capillary electrophoresis using positive charges to ethidium bromide intercalating agent that fluoresces
concentrate the DNA at the end of the capillary under UV light when bound to double-stranded
electropherogram data output from capillary DNA
electrophoresis where fluorescent signals are euploid having the proper number of chromosomes
recorded as graphical peaks per nucleus
electrophoresis separation of particles through a exclusion in human identification, difference of at
solution or matrix under the force of an electric least one allele from a reference source
current
exon sequences of DNA that code for protein
electrophoretic transfer movement of nucleic acid
exonuclease an enzyme that removes nucleotides
or protein from a gel matrix to a membrane using an
from DNA or RNA from either end of the linear
electric current
molecule
end labeling incorporation of radioactive or otherwise
expression array an array hybridized to RNA in
labeled nucleotides on the 3′ or 5′ end of a nucleic
order to measure gene expression
acid
extended haplotype nine STR loci on the
endonuclease an enzyme that separates DNA or RNA
Y chromosome, including the eight minimal
by cutting within the linear molecule
haplotype loci plus the highly polymorphic YCII
endpoint analysis analysis or observation of a PCR locus; used for lineage testing and matching
product after the amplification program is complete
extended MHC locus xMHC; an 8-Mb region of
engraftment establishment or repopulation of bone chromosome 6p including areas flanking the main
marrow cell lineages from a transplanted cell MHC locus
preparation
extracellular outside of the cell
enzyme induction stimulation of synthesis of
RNA-encoding enzymes or other factors from
inducible genes F
enzyme repression active prevention of the synthesis F factor a plasmid carrying instructions for the
of RNA-encoding enzymes or other factors from physical association of cells and transfer of genetic
inducible genes information from cell to cell
epidemic a disease or condition that affects many factor V Leiden a specific mutation in the gene
unrelated individuals at the same time coding for the factor V coagulation protein
epigenetics mitotically and meiotically heritable (F5 1691 A → G) resulting in the substitution of
changes in phenotype not encoded in genotype arginine at amino acid position 506 with glutamine
episome genetic material within the cell not attached false negative failure to detect an analyte present in a
to the host chromosome(s) test sample; analogous to Type II or beta error
epitope specific antigenic sites on a protein false positive results suggesting the presence of an
Eppendorf tube plastic conical tubes with 0.5- to analyte that is not in a test sample; analogous to
2.0-mL capacity Type I or alpha error
ERIC enterobacterial repetitive intergenic consensus; fidelity replication of template sequences without
palindromic sequences found in gram-negative errors
Glossary 513
field inversion gel electrophoresis (FIGE) pulsed gel matrix formed from large hydrated polymers with
field gel separation that uses alternation of positive variably sized spaces through which molecules can
and negative electrodes to resolve large particles move
filtering selection of variants found in a sequence gel mobility shift assay a method used to detect
based on variant features such as variant frequency, specific peptides by changes in electrophoretic
coverage, exon or intron location, germline/somatic migration speed upon binding to specific
antibodies
status, or other properties
gene an order or sequence of nucleotides on a
fragment analysis tests based on fragment sizes
chromosome that contains all the genetic information
determined by electrophoretic migration
to make a functional protein or RNA product
frameshift insertion or deletion in the nucleotide gene expression production of RNA and protein
sequence that throws the triplet code out of frame using a DNA template
framework region FR; a domain in the gene rearrangement intrachromosomal deletion and
immunoglobulin heavy-chain gene variable region ligation of gene segments in immunoglobulin and
coding for amino acids that do not directly contact T-cell receptor genes
antigen genetic code the relationship between DNA and
FRET fluorescent resonance energy transfer; a paired amino acid sequence
probe detection system for real-time PCR genetic concordance all alleles from two different
full chimerism complete replacement of recipient sources are the same
bone marrow with donor bone marrow cells after a genome all of the genes in an organism
transplant genomic imprinting enzymatic addition of methyl
fungicidal having the ability to destroy fungi groups to specific nitrogen bases in a predicted
pattern throughout the genome
fungistatic having the ability to inhibit the growth
and reproduction of fungi without killing them genotype the genetic DNA composition of an
organism
fusion gene a chimeric gene containing parts of two
germline having an innate, unmutated, or
or more separate genes
unrearranged genotype or genetic structure
gonadal mosaicism the presence of more than one
G genotype in the germ cells of an individual
G bands banding patterns of chromosomes after gradient gel gels with a range of concentrations of
staining with Giemsa urea and polyacrylamide
gain-of-function phenotype having a new undesired graft failure inability to detect donor bone marrow
trait; the new properties of the mutant allele are cells in a recipient after a bone marrow transplant
responsible for the phenotype even in the presence graft-versus-host disease GVHD; phenotypic
of the normal allele manifestations of immune reaction by donor cells
(graft) to the recipient (host) after a bone marrow
gamete a haploid reproductive cell
transplant
gap discontinuity in one strand of a double-stranded graft-versus-tumor effect GVT; immune reaction
nucleic acid; introduction of spacing used in by donor cells (graft) on residual tumor cells after a
sequence alignment bone marrow transplant for treatment of malignant
GC clamp DNA sequences rich in guanosine and disease
cytosine nucleotides that are more difficult to guanine one of the common nitrogen bases in DNA
denature into single strands and RNA
514 Glossary
guanosine one of the common nucleosides in DNA heterophagy intracellular degradation of extracellular
and RNA substrates taken into the cell by phagocytosis or
endocytosis
H heteroplasmy mitochondria with different sequences
in the same cell
hairpin a fold in DNA or RNA that forms a short
heterozygous in diploid organisms, having different
double strand along the single strand
alleles on homologous chromosomes
haploid having one of each chromosome
hierarchical sequencing determination of nucleotide
haplotype a genetically linked set of alleles that are order directed to known regions of the genome
inherited together
high-density oligonucleotide array a large number
haplotype diversity HD; calculated from the of probes (more than 100,000) synthesized in place
frequency of a given haplotype in a defined on the substrate
population
high-resolution banding staining of less compacted
hapten a small molecule capable of causing an chromosomes to produce a more detailed banding
immune response when attached to a larger (carrier) pattern
molecule histocompatible having the same alleles of the
Hardy–Weinberg equilibrium the relative frequency MHC locus
of two alleles in a population is constant histone a basic protein that associates with DNA
helicase an enzyme that untangles DNA by cutting histone code the relationship between histone
and ligating one or both strands of the double helix modification (methylation, acetylation,
hematopoietic stem cells HPC; undifferentiated phosphorylation) and control of gene expression
white blood cells that can differentiate into multiple HLA type collection of alleles in the major
blood cell lineages histocompatibility locus coding for different human
hemizygous presence of only one of two possible leukocyte antigens and detected by genotypic or
alleles in a diploid genotype phenotypic methods
heteroduplex double-stranded DNA in which Hoechst 33258 2′-[4-Hydroxyphenyl]-5-[4-
the component strands are not completely methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole
complementary trihydrochloride pentahydrate; DNA-specific dye
heteroduplex analysis HDA; detection of sequence used in fluorometric quantification of DNA
differences by denaturation and renaturation of test holoenzyme a multisubunit protein with enzymatic
and reference double-stranded nucleic acids, forming function
heteroduplexes where test sequences differ from home-brew describing a test system developed and
reference sequences validated within the clinical laboratory
heterologous noncomplementary; also describes homoduplex double-stranded DNA in which
two double-stranded nucleic acids with different the component strands are completely
nucleotide sequences complementary
heterologous extrinsic control nontarget templates homologous complementary; also describes two
added to a sample before amplification to ensure double-stranded nucleic acids with the same
proper sample purification and amplification nucleotide sequence
heterologous intrinsic control nontarget templates homologous extrinsic control a PCR template with
naturally occurring in a sample used to ensure primer-binding sites matching test targets and a
proper sample purification and amplification nontarget insert
Glossary 515
homoplasmy all mitochondria in a cell having the immobilized in molecular biology, bound to a
same sequences substrate such as a nitrocellulose-based membrane or
homozygous in diploid organisms, having the same glass slide
allele on both homologous chromosomes imprinting marking of DNA by the enzymatic
hot-start PCR preparation of PCR reaction mixes addition of methyl groups to specific nitrogen bases
so that no enzyme activity is possible until after the in silico analysis performed on a computer or by
mixes have gone through an initial heating in the computer simulation
thermal cycler in vitro analytical tests IVATs; reagent sets approved
HPLC high-performance liquid chromatography or by the FDA only for the detection of specific
high-pressure liquid chromatography; separation of analytes with no claim to clinical utility
components of a mixture in solution using a column in vitro diagnostic IVD; reagent sets intended for use
packed with stationary-phase material packed at high in the diagnosis of specific diseases; these may be
pressure FDA-approved tests
human leukocyte antigens HLA; gene products of inclusion in human identification, concordance of
the major histocompatibility locus in humans alleles with a reference source
humoral sensitization development of anti-human indel an insertion in DNA with a concomitant
antibodies, often due to organ transplant, blood deletion
transfusion, or pregnancy
indexing attachment of short (6-8 base) sample-
hybrid a product of genetically unrelated parents specific DNA sequences to sequencing templates
hybrid capture an ELISA-like assay using antibodies allowing multiple samples to be sequenced together;
to an RNA:DNA hybrid formed by hybridization of also called bar coding
target RNA with a DNA probe inducible transcription that is regulated by proteins
hybrid resistance rejection of transplanted organs by and other factors
immunocompromised mice informative locus a genetic locus that differs between
hybrid vigor survival advantage observed in two individuals
offspring of genetically unrelated parents over insertion gain or duplication of genetic material
offspring of genetically similar parents
internal control an analytical target distinguishable
hybridization in nucleic acids, the formation of from the test target but detectable with test reagents
hydrogen bonds between complementary strands of that is included in the reaction mix with the test
DNA or RNA target
hybridoma hybrid cells, often used to make internal labeling incorporation of radioactive
monoclonal antibodies nucleotides in chain termination sequencing for
hydrogen bond shared electrons between hydrogen visualization of the sequencing ladder
atoms; the basis of base pairing in nucleic acids internal transcribed spacer ITS; conserved elements
hypervariable region HV; a region found to found in regions separating the ribosomal RNA
have many different alleles (sequences) in a genes; used for typing yeast and molds
population; HV I and HV II are two such regions in interphase cell state in between mitosis or meiosis
mitochondrial DNA
interphase FISH cytogenetic probe analysis of
mitotic nuclei
I intracellular inside of the cell
iatrogenic infection caused by the actions of a intron sequences in DNA that interrupt coding
physician sequences
516 Glossary
Invader assay mutation/polymorphism detection L

using sequence-specific probes and a proprietary
laboratory-developed tests LDT; reagent sets and
cleavase enzyme
procedures validated in individual laboratories and
inversion intrachromosomal breakage, reversal, and not subject to special controls by the FDA
reunion of genetic material
lagging strand one strand of the parent double helix
inversion probe mutation/polymorphism detection that is read 5′ to 3′ and replicated discontinuously
method based on sequence-specific probe
hybridization, extension, ligation, and leading strand one strand of the parent double helix
amplification that is read 3′ to 5′ and replicated continuously
investigational use only IUO; reagents not intended leucine zipper specialized secondary structure found
for use on patient samples in proteins that bind DNA with leucine residues at
each seventh position for 30 residues
isochromosome chromosome containing two copies
of the same arm and loss of the other arm leukemia neoplastic disease of blood in which large
numbers of white blood cells populate the bone
isocratic a mobile phase in chromatography that
marrow and peripheral blood
remains at constant concentration throughout the
column leukocyte receptor cluster a group of genes on the
long arm of chromosome 19 coding for the killer
isodecoders transfer RNAs sharing anticodon
cell immunoglobulin-like receptors
sequences but differing in the sequences outside of
the anticodon library a set of short DNA fragments, prepared
from genomic DNA by nuclease digestion and
isotype a protein related to another through common
amplification or amplification with multiple PCR
function or structure; immunoglobulin classes
primers
directed against the same antigen
ligase an enzyme that forms one phosphodiester bond
connecting two preexisting fragments of DNA
K
likelihood ratio comparison of the probability that
kappa-deleting element KDE; a DNA sequence that two genotypes come from the same source with the
determines deletion of the IgK constant region in probability that they come from different sources
cells producing the IgL light chains limit of detection detection limit; lower limit of
karyotype the complete set of metaphase detection; the lowest target concentration that can be
chromosomes of a cell detected 95% of the time in a test assay
killer cell immunoglobulin-like receptor KIR; a linearity quantitative correlation between test result
protein expressed on the surface of NK cells and and actual amount of analyte
some memory T cells that interacts with HLA
linkage analysis use of alleles or phenotypes from
receptors
known locations in the genome to map genes
kinetochore a protein structure that attaches the
linkage disequilibrium the assumption that two
centromeres of chromosomes to the spindle
alleles are genetically linked
apparatus during cell division
linkage equilibrium the assumption that two alleles
kinship index likelihood ratio generated by a
are not genetically linked
determination of whether two people share one or
both parents; also called sibling index, combined linker region DNA between histones
sibling index locked nucleic acid LNA; nucleic acid with modified
Klenow fragment the large fragment of DNA sugar-phosphate backbone
polymerase without its exonuclease activity locus a defined site or location in a genome
Glossary 517
locus genotype the set of inherited alleles at a marker a site (gene or polymorphism) of known
particular site on homologous chromosomes, such as location in a genome
STR repeats or SNPs marker chromosome an unknown chromosome or
locus-specific brackets definition of a high and low part of a chromosome of unknown origin
limit of migration within which alleles are identified master mix preassembled components of a reaction
with a given degree of certainty solution
locus-specific RFLP detection of polymorphisms in match identity or inheritance of one or a set of
restriction sites within designated regions or genes; genetic alleles
used for typing of microorganisms
match probability probability of identity or
logit analysis a statistical method intended to inheritance of a set of genetic alleles
translate a quantifiable measure to a predicted state
matched unrelated donor MUD; a donor selected
or event (e.g., level of gene expression and clinical
from a pool based on HLA typing for a transplant
state)
Maxam–Gilbert sequencing a chemical sequencing
long interspersed nucleotide sequences LINEs;
method based on controlled breakage of DNA
highly repeated DNA sequences of 6 to 8 kbp in
length located throughout the human genome mecA The gene encoding the altered penicillin-
binding protein, PBP2a or PBP2′, which has a low
loss-of-function phenotype lacking a desired trait; in
binding affinity for methicillin
diploid organisms, inactivation of the normal allele
is responsible for the phenotype meiosis replication and separation of DNA by
reductional and equational divisions in diploid
loss of heterozygosity LOH; deletion or inactivation
organisms, resulting in four haploid products
of a functional allele, leaving a mutated allele
melt-curve analysis mutation/polymorphism scanning
lymphoma neoplasm of lymphocytes, capable of
based on changes in hybridization temperature
forming discrete tissue masses
melting temperature Tm; the temperature at which
Lyon hypothesis only one X chromosome remains
DNA is 50% single stranded and 50% double
genetically active in females; also called Lyon’s
stranded
hypothesis
messenger RNA ribonucleic acid that carries
lysosome cellular organelle in which cellular products
information from DNA in the cell nucleus to
are degraded
ribosomes in the cytoplasm
metacentric having the centromere in the middle of
M the chromosome
M13 a single-stranded DNA bacteriophage used in metaphase mitosis or meiosis phase where replicated
early procedures to make single-stranded templates compacted chromosomes align and prepare to
for sequence analysis separate
M13 universal primer a sequencing primer that metaphase FISH cytogenetic probe analysis of
could be used for all sequences cloned into the metaphase chromosomes
M13 RF plasmid metastasis movement of tumor cells from the original
macroarray a membrane containing multiple (primary) site of the tumor to other locations
immobilized probes microarray a small glass slide or other substrate
major groove the larger cleft in the double-stranded containing multiple immobilized probes
DNA helix microdeletion loss of a small amount of genetic
major histocompatibility locus MHC; a group of material barely detectable or undetectable by
genes located on chromosome 6p in humans karyotyping
518 Glossary
microelectronic array a small substrate containing mixed chimerism partial replacement of recipient
multiple immobilized probes subject to electrodes at bone marrow with donor bone marrow cells after a
each position on the array transplant
microfuge a small centrifuge designed to mixed lymphocyte culture MLC; measurement
accommodate 0.2- to 2.0-mL tubes of growth of lymphocytes activated by serum
microsatellite DNA from highly repeated short antibodies as an indication of donor/recipient
sequences (STRs) such that it differs in density from incompatibility; co-culturing of lymphocytes from
nonrepeated DNA unrelated individuals to determine HLA type
microsatellite instability MSI; contraction and mobility capacity for migration
expansion of mononucleotide and dinucleotide Molecular Beacons a self-annealing probe detection
repeat sequences in DNA caused by lack or repair of system used in real-time PCR
replication errors monoclonal (monotypic) having the same genotypic
microvariant a repeated sequence in DNA missing or phenotypic composition
part of the repeat unit
monoclonal antibody antibody preparation designed
migration in electrophoresis, movement of particles to recognize a defined antigen or epitope
through a matrix under the force of a current
monomer a single protein or peptide that can
minimal haplotype eight Y-STR loci used for function alone
paternal lineage testing and matching
monosomy in diploid organisms, loss of a single
minimum inhibitory concentration MIC; the least chromosome
amount of antimicrobial agent that inhibits the
mosaic an individual harboring genotypically
growth of an organism
different cells or tissue arising from a single zygote
minisatellite DNA from highly repeated sequences
MRSA methicillin-resistant Staphylococcus aureus;
(VNTRs) such that it differs in density from
a strain of S. aureus that has resistance to multiple
nonrepeated DNA
antibiotics
mini-STR a system of PCR amplification of an STR
multilocus sequence typing MLST; characterization
locus using primers that produce a smaller amplicon
of bacteria using sequences of internal fragments of
than standard STR systems
housekeeping genes
minor groove the smaller cleft in the double-stranded
DNA helix multiple-locus probe MLP; a Southern blot probe set
that hybridizes to more than one locus on the same
minor groove binding MGB; the nature of blot
association of a molecule with double-stranded DNA
within the minor groove of the double helix multiplex PCR amplification of more than one target
in a single PCR reaction
minor histocompatibility antigens mHags; proteins
outside of the main MHC locus that affect organ mutation a change in the order or sequence of
engraftment nucleotides in DNA found in less than 1% to 2% of
a given population
misprime aberrant initiation of DNA synthesis from
a primer hybridized to template sequences different myeloablative in bone marrow transplants, removal
from the intended target of recipient bone marrow
mitochondrion a subcellular organelle responsible for
energy production in the cell N
mitogen an agent that promotes cell division NASBA nucleic acid sequence–based amplification;
mitosis separation of DNA in diploid organisms by an isothermal DNA amplification process that
equational division, resulting in two diploid products includes an RNA intermediate
Glossary 519
necrosis cell and tissue destruction upon cell death nonsense substitution an alteration in the nucleotide
negative control an analytical target that does sequence that results in the substitution of a
not react with reagent/detection systems; used to termination codon for an amino acid codon
demonstrate that a procedure is functioning with northern blot a method used to analyze specific RNA
proper specificity and without contamination transcripts in a mixture of other RNA
negative template control template sequences that do nosocomial hospital-caused infection
not match the intended target NTP nucleotide triphosphate
neoplasm growth of tissue that exceeds and is not nucleic acid a linear polymer of nucleotides
coordinated with normal tissue; a tumor
nucleic acid sequence-based amplification NASBA:
nested PCR amplification of the same target in replication of a DNA or RNA target through an
two PCR reactions, using the product of the first intermediate RNA product
amplification as the template for a second round of
nuclein viscous substance isolated from nuclei by
amplification with primers designed to hybridize
Miescher, later to be known as DNA
within the amplicon formed by the first primer set
nucleoside a unit of nucleic acid comprised of a
new mutation a spontaneous mutation arising in
ribose sugar and a nitrogen base
germ cells of an unaffected individual
nucleosome eight histones wrapped in approximately
nick translation addition of labeled nucleotides at
150 bp of DNA
nicks (single-strand breaks) in DNA
nucleotide a unit of nucleic acid comprising a
nitrocellulose matrix material with high binding
phosphorylated ribose sugar and a nitrogen base
capacity for nucleic acids and proteins
null allele a genetic type or variant with no
nitrogen base a carbon-nitrogen ring structure that
phenotypic effect
comprises part of a nucleoside
nonconservative substitution an alteration in the
nucleotide sequence that results in the substitution of O
an amino acid with one of dissimilar properties Okazaki fragments intermediate products of DNA
nondisjunction abnormal separation of chromosomes synthesis on the lagging strand
during cell division resulting in both of a oligoclone a small subpopulation with identical
chromosome pair in one daughter cell genotype or phenotype within a larger group
nonhistone proteins chromosome-associated proteins oligomer proteins or peptides that function as
that maintain chromosomal compaction in the components of more than one unit (dimer, trimer,
nucleus tetramer); in nucleic acids, a short sequence of
noninformative locus a genetic locus that is identical nucleic acid
in two individuals oligonucleotide array a small glass slide or other
nonisotope RNase cleavage assay NIRCA; a substrate containing multiple immobilized probes
mutation/polymorphism screening and amplification synthesized directly on the substrate; high-density
method using RNase cleavage of heteroduplexes oligonucleotide arrays
nonpolar hydrophobic; water insoluble oncogene a mutated gene that promotes the
nonsense codon a trinucleotide sequence that proliferation and survival of cancer cells
terminates translation; UAA, UAG, UGA oncology the study of cancer
nonsense-mediated decay degradation of mRNA oncomir a microRNA that acts as a tumor-suppressor
with premature stop codons gene or an oncogene
520 Glossary
ontogeny the course of development, for example, of phenotype the biological properties of an organism
an individual organism phosphodiester bond covalent attachment of the
Oxford sequence sequence of the mitochondrial hydroxyl oxygen of one phosphorylated ribose (or
hypervariable regions used as a reference for deoxyribose) sugar to the phosphate phosphorous of
defining polymorphisms the next
phosphor a substance that glows after exposure to
P electrons or ultraviolet light
photobleaching fading; photochemical destruction of
p in cytogenetics, indicating the short arm of a
a fluorescent molecule (fluorophore) by exposure to
chromosome
light
P site peptidyl site, location of charged tRNA binding
Phrap a sequence assembly tool
in the ribosome complex
PAM protospacer adjacent motif; a short DNA Phred a sequence quality assessment system
sequence required for recognition and cutting of phylogeny history and evolutionary development of a
target sequences by Cas nucleases species or related group of organisms
pandemic a disease or condition found across wide plasma cells mature white blood cells that produce
geographical areas at the same time antibodies
panel reactive antibodies PRA; a set of reference plasmid small, usually circular double-stranded DNA,
antibodies used to define the HLA of reference often carrying genetic information, that replicates
lymphocytes independently or in synchrony with host cell
replication
paracentric inversion intrachromosomal breakage,
reversal, and reunion of genetic material, not plasmid fingerprinting isolation and restriction
including the centromere mapping of bacterial plasmid for epidemiological
studies
paternity index an expression of how many times
more likely it is that a child’s allele is inherited from point mutation DNA sequence alteration involving
an alleged father than from a random man in the one or a few base pairs
population polar hydrophilic; water soluble
pedigree a diagram of the inheritance pattern of a polarity orientation of nucleic acid such that one end
phenotype (or genotype) in a family (5′) has a phosphate group and the other end (3′) has
a hydroxyl group and is the site of extension of the
penetrance frequency of expression of a disease
molecule
phenotype in individuals with a gene lesion
polony a cluster of PCR products primed by surface-
peptide a polymer of a few amino acids
attached oligonucleotides
peptide bond covalent carbon-nitrogen bonds that
polyA tail polyadenylic acid at the 3′ terminus of
connect amino acids in proteins
messenger RNA
peptide nucleic acid PNA, nucleic acid with peptide polyacrylamide polymer of acrylamide and
bonds replacing the sugar-phosphate backbone bis-acrylamide that, when hydrated, forms a gel
peptidyl transferase ribozyme that catalyzes the frequently used for sieving proteins and nucleic
formation of a peptide bond acids
pericentric inversion intrachromosomal breakage, polyadenylate polymerase the enzyme that catalyzes
reversal, and reunion of genetic material, including the formation of polyadenylic acid at the 3′ terminus
the centromere of messenger RNA
pH drift decrease of pH at the cathode and increase polyadenylation addition of adenosines to the 3′ end
in pH at the anode during electrophoresis of a messenger RNA molecule
Glossary 521
polyadenylation signal the site of activation of premutation a genetic lesion not manifesting a
termination in eukaryotic RNA synthesis disease phenotype but prone to advance to full
polyadenylic acid nucleic acid comprising only mutation/disease status
adenosine nucleotides pre-PCR laboratory areas and materials not exposed
polyclonal (polytypic) having a variety of genotypes to PCR products
or phenotypes preventive maintenance routine review of instrument
polyclonal antibody a collection of antibodies function with appropriate part replacement
produced in vivo that recognizes an antigen or primary antibody antibody that directly binds a
epitope target molecule
polymerase in nucleic acids, an enzyme that primary resistance mutation mutations that are
connects nucleotides by catalyzing the formation of specific for a given antibiotic or antiviral drug
phosphodiester bonds and make an organism less susceptible to that
polymerase chain reaction PCR; primer-directed drug
DNA polymerization in vitro resulting in primary structure in proteins, the sequence of
amplification of specific DNA sequences the amino acids; in nucleic acids, the sequence of
polymorphism a difference in DNA sequence found nucleotides
in 1% to 2% or more of a given population primase the enzyme that produces short (~60 bp)
complementary RNAs to prime DNA synthesis
polypeptide protein
primer a short, single-stranded DNA fragment
polyphred a sequence quality assessment designed to
designed to primer synthesis of a specific DNA
detect single-nucleotide changes region
polyploidy in diploid organisms, having more than primer dimer a side product of PCR resulting from
two chromosome complements amplification of primers caused by 3′ homology in
polysomy in diploid organisms, having more than two primer sequences
of a single chromosome prion aberrantly folded proteins capable of
polythymine nucleic acid comprising only thymine transferring their configuration to normal proteins
nucleotides; poly T prior odds initial probability of an event in the
polyuracil nucleic acid comprising only uracil absence of a test effect
nucleotides; poly U private antibodies HLA type-specific antibodies
position effect differences in gene transcription probability of paternity a number calculated from
depending on chromosomal location the combined paternity index and reported in
paternity testing
positive control an analytical target known to react
with reagent/detection systems; used to demonstrate proband the initial patient resulting in a genetic study
that a procedure is functioning properly of a family
post-PCR laboratory areas and materials used for probe in blotting procedures, nucleic acid or protein
with a detectable signal that specifically binds to
working with PCR products
complementary sequences or target protein
preanalytical error events occurring prior to sample
probe amplification an approach to increase the
analysis that adversely affect test results
sensitivity of target detection by protection and
precision reproducibility of test results amplification of the probe, rather than the target
prehybridization treatment of membranes before probit analysis a statistical method to form binary
introduction of probe to minimize nonspecific groups based on quantitative data; used to determine
binding cut points for qualitative tests
522 Glossary
processivity the ability of polymerase to stay with the pulsed-field gel electrophoresis PFGE, a technique
template during replication of long sequences using alternating currents to move very large DNA
product rule the frequency of a genotype in a fragments through an agarose gel
population is the product of the frequencies of its purine nitrogen bases with a double-ring structure;
component alleles adenine and guanine are purines
proficiency testing analysis of specimens supplied to pyrimidine nitrogen bases with a single-ring
independent laboratories from an external reference structure; cytosine, thymine, and uracil are
source pyrimidines
profile a set of markers, alleles, or other pyrimidine dimers boxy structures resulting from
characteristics in an individual covalent attachment of cytosine or thymine with an
promoter DNA sequences that bind RNA polymerase adjacent thymine in the same DNA strand; formed
and associated factors when DNA is exposed to UV light
proofreading a correction function of the DNA pyrogram luminescence data output from a
polymerase complex that repairs errors committed pyrosequencer
during DNA replication pyrophosphate an ester of pyrophosphoric acid
prophase early mitosis or meiosis containing two phosphorous atoms, P2O74−
prosthetic group nonprotein moiety, such as a sugar pyrosequencing a sequencing method based on the
or lipid group covalently attached to a protein release of pyrophosphates during DNA replication
protease an enzyme that dissembles protein into its
component amino acids Q
protein a polymer of amino acids with structural or q in cytogenetics, indicating the long arm of a
functional capabilities chromosome
protein truncation test mutation/polymorphism Q banding banding patterns of chromosomes after
analysis using in vitro transcription–translation staining with quinacrine fluorescent dyes
systems Qβ replicase RNA-dependent RNA polymerase from
proteome all of the proteins that make up a cell the bacteriophage Qβ
proteomics study of the proteome, usually through quantitative PCR qPCR, real-time PCR
array or mass spectrometry analysis quaternary structure functional association of
proto-oncogene a normal gene with the potential separate proteins or peptides
to promote aberrant survival and proliferation if quencher a molecule that takes fluorescent energy
mutated from a reporter dye
protospacer the part of the CRISPR crRNA sequence
that is complementary to the target R
pseudoautosomal describing homologous regions R banding modified G banding using acridine
in the X and Y chromosomes that undergo crossing orange; the resulting pattern will be opposite of that
over during meiosis of G banding
pseudogene intron-less, nonfunctional copies of R loop a hybrid structure formed between RNA and
genes in DNA DNA such that the unpaired introns in the DNA
psoralen(s) a substance from plants that covalently form loops
attaches to DNA under ultraviolet light random priming template-directed synthesis
public antibodies antibodies that recognize more of single strands of DNA carrying labeled
than one HLA type nucleotides
Glossary 523
randomly amplified polymorphic DNA RAPD; reporter dye a fluorescent dye whose signal indicates
a PCR method using random priming with 6- to the presence of target
10-base primers for typing of organisms; also known REP-PCR repetitive extragenic palindromic PCR;
as arbitrarily primed PCR analysis of amplicons produced from primers
reagent alcohol a mixture of 90.25% ethanol, homologous to interspersed recurring sequences in
4.75% methanol, and 5% isopropanol microorganisms
reagent blank PCR reaction mix with all components reproducible consistency of test results produced
except template from the same procedure
real-time PCR PCR performed in an instrument that research use only RUO; reagents not intended for
can measure the formation of amplicons in real time; use on patient samples
also called quantitative PCR (qPCR) resequencing direct sequence analysis of the same
recessive Mendelian inheritance pattern where area in multiple samples to detect variants; usually
offspring have a 25% likelihood of expressing an applied to known mutations or areas of frequent
affected parental phenotype mutation
reciprocal translocation exchange of DNA between resolution the level of detail at which an allele is
separate chromosomes with no gain or loss of determined; also, the separation of particles by
genetic material electrophoresis or chromatography
recognition site sequences in DNA that are bound by restriction endonuclease an enzyme that makes
proteins such as restriction enzymes or transcription double-stranded cuts in DNA at specific sequences
factors
restriction enzyme an endonuclease isolated from
recombinant a new combination of DNA sequences bacteria that will break single- or double-stranded
that is produced from different preexisting (parent) nucleic acids at specific sequences
sequences; a cell or organism containing a new
restriction fragment length polymorphism RFLP;
combination of sequences
a sequence variation that results in creating,
recombine to mix different DNA sequences such that destroying, or moving a restriction site
a new combination of sequences is produced from
restriction map a diagram showing the linear
different preexisting (parent) sequences
placement of restriction enzyme recognition sites in
redundant tiling design of probes on an array such DNA
that a predictable mutation is located at different
places in the probe sequence reverse dot blot hybridization technique with
multiple probes spotted on a membrane all exposed
reference range expected analyte frequency or levels to the same target
from a population of individuals
reverse transcriptase an RNA-dependent DNA
REF-SSCP mutation/polymorphism scanning by polymerase
SSCP in combination with restriction enzyme
fingerprinting; see single-strand conformation reverse-transcriptase PCR RT-PCR; polymerase
polymorphism chain reaction amplifying cDNA made from RNA by
reverse transcriptase
regulatory sequence that part of a gene sequence that
binds factors controlling the expression of the gene RF part of the M13 life cycle, where its genome is in
the form of a double-stranded DNA circle
release factor specialized protein required for the
dissociation of the translation complex at the end of ribonuclease an enzyme that separates RNA into its
protein synthesis component nucleotides
reportable range the range within which test results ribonucleic acid RNA; a polymer of ribonucleotides
are considered to be valid (with or without dilution) ribose a five-carbon sugar
524 Glossary
ribosomal binding site mRNA sequence that binds to Sanger sequencing dideoxy chain termination
ribosomes in preparation for protein synthesis sequencing
ribosomal RNA ribonucleic acid that serves as a sarcoma tumor of bone, cartilage, muscle, blood
structural and functional component of ribosomes vessels, or fat
ribosome complex of RNA and proteins that Scorpion a self-annealing labeled primer detection
catalyzes formation of peptide bonds system for real-time PCR
ribotyping typing of microorganisms using restriction
secondary antibody antibody that binds primary
fragment polymorphisms in ribosomal RNA genes
antibody to a target molecule
ribozyme RNA with enzymatic activity
secondary resistance mutation secondary mutations
ring chromosome a circular structure formed
that enable an organism with primary resistance
from the fusion of the ends of a chromosome or
mutations to replicate in the presence of a given
chromosome fragment
antibiotic or antiviral drug
RNA integrity control an amplification control
included in RT-PCR to distinguish negative and secondary structure specific folding of protein or
false-negative results nucleic acid through the interaction of amino acid
RNA-dependent RNA polymerases enzymes that side chains or nitrogen bases, respectively
synthesizes RNA using RNA as a template selenocysteine cysteine with selenium replacing the
RNA polymerase an enzyme that catalyzes the sulfur atom; the 21st amino acid
template-dependent formation of phosphodiester selenoprotein a protein containing selenocysteine
bonds between ribonucleotides forming ribonucleic self-sustaining sequence replication SSSR or 3SR;
acid replication of a DNA or RNA target through an
RNAse-free (RNF) reagents, consumables, and intermediate RNA product
facilities prepared or treated to protect RNA from
semi-conservative a term used to describe DNA
RNA digesting enzymes
replication where one strand is conserved and
RNase H an exonuclease that digests RNA from serves as a template for a new strand, resulting in
RNA:DNA hybrids a new double helix comprising one parent and one
RNase protection a method used to detect transcripts daughter strand
by hybridizing test RNA with a labeled probe and
semi-nested PCR amplification of the same target
removing un-hybridized target and probe with
in two PCR reactions, using the product of the first
single-strand-specific nucleus
amplification as the template for a second round of
RNA-SSCP rSSCP; mutation/polymorphism scanning amplification with primers, one of which is the same
using RNA instead of DNA; see single-strand as the first round and one of which is designed to
conformation polymorphism hybridize within the amplicon formed by the first
primer set
S sense strand the single strand of a DNA double helix
S sedimentation coefficient; used to describe the that is not used as a template for messenger RNA
density (movement) of particles in a given medium synthesis
under gravity or centrifugal force sensitivity control an analytical target known to react
S1 analysis a method used to detect transcripts by with reagent/detection systems but present in the
hybridizing test RNA with a labeled probe and lowest detectable concentration; used to demonstrate
removing un-hybridized target and probe with that the procedure is detecting target down to the
single-strand-specific nucleus indicated input levels of testing
salting out purification of nucleic acid by sequence the order of nucleotides in a nucleic acid
precipitating proteins and other contaminants with polymer; the order of amino acids in a protein
high salt at low pH polymer
Glossary 525
sequence-based typing SBT; determination of HLA signal transduction transfer of extracellular and
DNA polymorphisms by direct sequencing intracellular stimuli through the cell cytoplasm to
sequence complexity the length of nonrepetitive the nucleus, ultimately affecting gene-expression
nucleic acid sequences patterns
sequence-specific oligonucleotide probe silent substitution an alteration in the nucleotide
hybridization SSOP, SSOPH; detection of sequence that does not change the amino acid
DNA polymorphisms using amplified test DNA sequence
immobilized on a filter exposed to labeled probes silver stain a sensitive staining system for nucleic
that will hybridize to specific sequences acids and proteins after gel electrophoresis
sequence-specific (primer) PCR SSP, SSP-PCR; single-locus probe a Southern blot probe that
detection of point mutations or polymorphisms in hybridizes to only one locus
DNA with primers designed so that the 3′-most single-nucleotide polymorphism SNP; a 1-bp
base of the primer hybridizes to the test nucleotide variation from a reference DNA sequence
position in the template single-strand conformation polymorphism SSCP;
sequencing ladder the pattern of sequence reaction a method designed to detect sequence alterations in
products on a polyacrylamide gel DNA through differences in secondary structure due
serology study of antigens or antibodies in serum to differences in the nucleotide sequence
sex-linked having genetic components located on the SISH silver-enhanced in situ hybridization; a bright-
X or Y chromosome field hybridization method similar to CISH for the
sgRNA synthetically fused crRNA and tracrRNA that detection of chromosomal abnormalities and gene
can be designed to target specific DNA sequences amplifications
for cutting with Cas9 endonuclease slot blot a membrane containing multiple targets
deposited in an elongated pattern rather than a “dot”
sharkstooth comb a specialized comb used to
generate lanes immediately adjacent to one another solution hybridization hybridization of nucleic acid
to facilitate lane-to-lane comparison target and labeled probe in solution
short interspersed nucleotide sequences SINEs; somatic hypermutation enzymatic alterations of
highly repeated sequences approximately 0.3 kbp in nucleotide sequences in the variable region of the
length located throughout the human genome immunoglobulin heavy-chain gene
Southern blot a method used to analyze specific
short tandem repeats STR; head-to-tail repeats of
regions of DNA in a mixture of other DNA
DNA sequences with less than 10-bp repeat units
regions
shotgun sequencing determination of nucleotide
spa typing tandem repeat elements in the 3′ coding
sequence by the assembly of sequence data from a
region of the Staphylococcus aureus protein A gene,
random collection of small fragments
spa, analyzed by PFGE to identify MRSA
sibling index likelihood ratio generated by a spectral karyotyping cytogenetic probe analysis
determination of whether two people share one or designed to visualize the entire complement of
both parents; also called kinship index or combined metaphase chromosomes, with unique colors for
sibling index each chromosome
side chain that portion of an amino acid with spin column small silica columns designed to fit
biochemical properties within an Eppendorf tube, using centrifugal force to
signal amplification an approach to increase the wash and elute bound substances
sensitivity of target detection by amplification of the splicing Removal of intron sequences from messenger
probe signal rather than the target RNA
526 Glossary
split chimerism differential replacement of specific synonymous describing DNA sequence variants that
recipient bone marrow cell lineages with donor bone do not change the reference amino acid sequence
marrow cells after a transplant
split specificity antigenic types derived from a parent T
antigen
tag SNP one or two single-nucleotide polymorphisms
standard curve graphical depiction of results
that can be used to define a haplotype or ~10,000-bp
from a dilution series of analyte encompassing
block of DNA sequences
levels predicted to result from unknown test
specimens Taq polymerase a heat-stable polymerase isolated
from Thermus aquaticus
standard precautions recommendations by the
Centers for Disease Control and Prevention for TaqMan a probe detection system used in real-time
handling potentially infectious specimens PCR
standard tiling design of probes on an array such target specific sequences or regions that are detected
that the mutation site is always in the same position in an analytical procedure
from the end of the probe target amplification replication of a specific target
Stoffel fragment Taq polymerase lacking region of DNA
289 N-terminal amino acids TE buffer 10-mM Tris, 1-mM EDTA buffer
stop codon nonsense codon telocentric having the centromere at one end of the
strand one molecule of nucleic acid; a double helix chromosome
contains two strands of DNA telomere the end of a chromosome; composed of
strand displacement amplification SDA; an repeated DNA sequences and associated proteins
isothermal amplification reaction using nick telomeric probe probes that bind to telomeres
translation and strand displacement
TEMED N,N,N′,N′-tetramethylethylenediamine,
structural sequence that part of a gene sequence that with APS, catalyzes polymerization of acrylamide
codes for amino acids gels
stutter a PCR artifact that results from slippage of template a region of single-stranded nucleic acid
the template on short repeated sequences
used as a guide for replication or synthesis of a
submarine gel agarose gels loaded under the buffer complementary strand
surface
teratocarcinoma tumors comprising multiple cell
submetacentric describing a chromosome with a types
centrally located centromere closer to one end of the
terminal transferase an enzyme that adds
chromosome than the other
nucleotides to the end of a strand of nucleic acid
subpopulation genetically defined groups within a
without using a template
population
tertiary structure configuration of folded proteins
sugar-phosphate backbone the chain of
required for function
phosphodiester bonds that hold nucleotides together
in a single strand of nucleic acid tetraploid having four of each chromosome
surname test a test of paternal lineage using alleles threshold cycle CT; in real-time PCR, the PCR cycle
on the Y chromosome at which the fluorescence from the PCR product
surpasses a set cut point
susceptibility testing detection of resistance to given
antimicrobial agents by the ability to grow in the thymidine one of the common nucleosides in DNA
presence of the agent and RNA
SYBR green a fluorescent dye specific to double- thymine one of the common nitrogen bases in DNA
stranded nucleic acid and RNA
Glossary 527
thymine dimers boxy structures resulting from transmission pattern mode of inheritance of a
covalent attachment of adjacent thymines in the phenotype within a family
same DNA strand; formed when DNA is exposed to transposon a fragment of DNA with the capacity to
UV light move from one genetic location to another
tissue typing identification of HLA types trimming addition or deletion of nucleotides
topoisomerase a helicase that untangles DNA by at the junctions between V, D, and J segments
cutting and ligating one or both strands of the during immunoglobulin and T-cell receptor gene
double helix rearrangements
touchdown PCR a modification of the polymerase trinucleotide repeat three 3-bp tandem sequence
chain reaction method to enhance amplification repeats in DNA
of desired targets by manipulation of annealing
triploid having three of each chromosome
temperatures
true negative lack of detection of a target marker that
tracking dye in electrophoresis, a dye added to
samples being separated to monitor the extent of is not present in a test sample
migration through the matrix true positive accurate detection of a target marker
tracrRNA noncoding RNA that forms a present in a test sample
ribonucleoprotein complex with crRNA and truncation premature protein termination
Cas9 endonuclease in bacteria to target invading Tth polymerase a heat-stable polymerase isolated
DNA from Thermus thermophilus
trait a particular characteristic tumor-suppressor gene TSG; a gene that dampens
transcription template-guided synthesis of RNA by proliferation and survival; TSG function is often lost
RNA polymerase in cancer cells
transcription-mediated amplification TMA; an typing capacity ability of a test to identify the target
isothermal amplification method using an RNA organism
intermediate; TMA is also an abbreviation for tissue
typing trays plates preloaded with reference
microarrays
antibodies used for study of HLA antigens
transduction transfer of genetic information from
tyrosine kinase inhibitor TKI; chemotherapeutic
one cell to another through a viral intermediate
agent that inhibits phosphorylation catalyzed by
transfer in blotting, movement of DNA or protein oncogenes such as EGFR and BCR-ABL
resolved by electrophoresis from the gel matrix to a
membrane
transfer RNA adaptors that link codons in messenger U
RNA with amino acids unbalanced in cytogenetics, genetic events that result
transformation transfer of genetic information in the gain or loss of genetic material
among cells without physical association, such that a
uniparental disomy inheritance of chromosomal
new phenotype is produced in the recipient cells
material from only one parent due to aberrant
translocation breakage and fusion of separate separation of chromosomes during meiosis
chromosomes
transmembrane spanning across the cell membrane
V
transmission-based precautions protective
equipment and/or clothing used with potential vacuum transfer movement of nucleic acid or
exposure to airborne or contact-transmissible protein from a gel matrix to a membrane using a
infectious agents vacuum
528 Glossary
validation a series of analyte tests using a particular whole chromosome paints fluorescent probes that
method to establish method characteristics stain the entire length of a specific chromosome
variable expressivity a range of phenotypes in
individuals with the same gene lesion X
variable-number tandem repeats VNTR; head-to-
tail repeats in DNA with 10- to 150-bp repeat units X-linked having genetic components located on the
V(D)J recombination normal intrachromosomal X chromosome
breaking and joining of DNA in the genes coding for
immunoglobulins and T-cell receptors Y
viral load quantified presence of virus in a specimen
Y-STR short tandem repeats located exclusively on
virus a microorganism dependent on host cell
the Y chromosome
machinery for reproduction; also, a sometimes-
deleterious electronic signal that can be passed Y-STR/paternal lineage test establishment of
among computers paternal ancestry using Y chromosome polymorphic
short tandem repeat haplotypes
W
Z
well in gel electrophoresis, an indentation or space
molded into the gel to accommodate loading of zinc finger specialized Zn-stabilized secondary
molecules to be separated structure found in proteins that bind DNA
western blot a method used to analyze specific zwitterion a molecule that can be completely
proteins or peptides in a mixture of other proteins positively or negatively charged at a given pH
Index
Numbers followed by f represent figures, numbers followed by t represent tables, and numbers followed by b represent boxes.
A Allele-specific oligomer hybridization (ASO), 209

A site, 49, 49f dot blot, 209
Absorptivity constants, 92 reverse dot blot, 209
Acrocentric chromosome, 184, 184f Allele-specific primer amplification, 214f
Acrylic shielding, 466, 467f Allelic discrimination with fluorogenic probes, 214–215, 215f
Activator, 63 Allelic exclusion, 388
Acute lymphocytic leukemia, 406t Allelic frequencies, paternity testing, 275–277
Acute monocytic leukemia, 406t combined paternity index, 276
Acute myeloid leukemia, 406t paternity index, 275–276
Acute myeloid leukemia/myelodysplastic syndrome, 406t prior odds, 276–277, 277t
Acute myelomonocytic leukemia, 406t probability of paternity, 276–277
Acute nonlymphocytic leukemia, 406t Allelic ladders, 268
Acute promyelocytic leukemia, 406t Alloantibodies, 428
Acute T-lymphocytic leukemia, 406t Allogeneic transplant, 283
Adaptors, 16 Allografts, 418
Adenine, 3 Alpha helix, protein, 42, 42f
Adenosine, 4 Alpha phosphate, 29, 59
Adler, Julius, 12b Alpha satellite, 183
Affinity maturation, 391b Alternative nucleic acids, 126b
AFLP analysis, 334f Alternative splicing, 33, 121–122
Agarobiose, 99f Alu elements, 261
Agarose, 98, 99f, 99t Ambiguity, 425
Agarose gel electrophoresis, 91f Amelogenin locus, 270, 270f
Agarose gels, 99–101 Amino acid, 38–43, 39f
Alanine tRNA, 34f Amino acid biosynthetic groups, 40t
Algorithm, 249t Amino acid charging, 46–47
Alignment, 249t Amino acid classification, 40t
Alignments of HLA polymorphic regions, 431 Amino acyl tRNA synthetases, 47f
Alkaline phosphatase, 132b, 201, 202b, 202f, 227b Amino-terminal, 38
Allele dropout, 242 Aminoacyl tRNA synthetases, 46
Allele peak locations, 269f Ammonium persulfate (APS), 102
Alleles Amplicons, 143, 147f
defined, 420 Amplification
HLA nomenclature, 421–422, 425 control, 153, 305, 306f, 457
HLA polymorphisms, 420 probe, 168–171
identification serologically and by DNA sequence, 426t program, 144
locus versions, 263 signal, 172–174
null allele, 425 target, 143–168
529
530 Index
Amplified fragment length polymorphism, 332–333 immunoglobulin light-chain gene re-arrangement in B cells,
Analyte measurement range, 454t 389–390
Analyte-specific reagents (ASR), 464 kappa deleting element, 389
Analytic accuracy, 454, 454t somatic hypermutation, 388
Analytic sensitivity, 454t T-cell receptor gene rearrangement, 390–391
Analytic specificity, 454t trimming, 391
Analytical targets, molecular testing, 372 leukemias, chromosomal abnormalities, 406t
acute lymphocytic leukemia, 406t liquid biopsy, 386–387
acute monocytic leukemia, 406t lymphomas, chromosomal abnormalities, 406t
acute myeloid leukemia, 406t mantle cell lymphoma, 406t
acute myeloid leukemia/myelodysplastic syndrome, 406t microsatellite instability, 382, 384–385, 385f
acute myelomonocytic leukemia, 406t molecular abnormalities, 382
acute nonlymphocytic leukemia, 406t mucosa-associated lymphoid tissue lymphoma, 406t
acute promyelocytic leukemia, 406t multiple myeloma, 406t
acute T-lymphocytic leukemia, 406t myeloproliferative/myeloblastic disease, 406t
anaplastic lymphoma receptor tyrosine kinase (ALK) proto- myeloproliferative/myelodysplastic disease, 406t
oncogene, 2p23.1, 382 neuroblastoma ras, N-ras (1p13), 375–376, 375f, 375t
aneuploidy, 372 paired box-forkhead in rhabdomyosarcoma, PAX3-FKHR, PAX7-
ataxia telangiectasia mutated gene, ATM (11q22), 379–380 FKHR, t(1;13), t(2;13), 378
B-cell leukemia, 406t polycythemia vera, 406t
breast cancer 1 gene, BRCA1 (17q21), and breast cancer 2 gene, pre-B acute lymphoblastic leukemia, 406t
BRCA2 (13q12), 380 proto-oncogenes, 381
Burkitt lymphoma, 406t rearranged during transfection proto-oncogene (10q11), 381
chronic lymphocytic leukemia, 406t rearranged during transfection (RET) proto-oncogene (10q11), 381
chronic myelogenous leukemia, 406t sequencing panels, 405
clonality detection, 391 synovial sarcoma translocation, chromosome 18-synovial
banding patterns, 396–397 sarcoma breakpoint 1 and 2, SYT-SSX1, SYT-SSX2 t(X;18)
complementarity determining regions, 393 (p11.2;q11.2), 377–378
consensus primer, 393 T-cell lymphoma, 406t
framework regions, 393 T-chronic lymphocytic leukemia, 406t
hematological malignancies, gene mutations, 404–405 tissue-specific, 372
immunoglobulin heavy chain gene rearrangements, tumor protein 53, TP53 (17p13), 378–379
391–394 tumor-specific, 372
immunoglobulin light chain gene rearrangements, 394 V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene
mutations, hematological malignancies, 397–405 homolog, Kit, c-KIT (4q12), 382
polyclonal lymphocytes, 391 V-myc avian myelocytomatosis viral-related oncogene,
T-cell receptor gene rearrangements, 394–396 neuroblastoma-derived, 381
diffuse large B-cell lymphoma, 406t Von Hippel-Lindau gene, VHL (3p26), 380–381
epidermal growth factor receptor, EGFR (7p12), 373–375 V-Ros Avian UR2 Sarcoma Virus Oncogene Homolog 1 (ROS1)
Ewing sarcoma, EWS (22q12), 376–377, 377t, 378f Proto-Oncogene (6q22.1), 381
follicular lymphoma, 406t Waldenström macroglobinemia, 406t
gene/chromosomal mutations, solid tumors, 372–387 Anaplastic lymphoma receptor tyrosine kinase (ALK) proto-oncogene,
hairy cell leukemia, 406t 2p23.1, 382
Harvey rat sarcoma viral oncogene homolog, H-ras (11p15), Anderson reference, 291
375–376, 375f, 375t Aneuploid cells, 181
hematological malignancies, molecular analysis, 387–406 Aneuploidy, 181, 187f, 188f, 345, 372
hematological malignancies, molecular targets Anion, 123
allelic exclusion, 388 Annealing, 145, 146f, 147b
gene rearrangements in leukemia and lymphoma, 387–397 Annotation, 246, 249t
germline, 387–388 Anode, 98, 120
immunoglobulin heavy-chain gene re-arrangement, B cells, Anticodon, 34, 45–47
387–388 Antigen detection, 202f
V(D)J recombination, 387, 387f Antigen retrieval, 202
heterozygosity loss, 385, 386f Antihuman antibodies, 428
human epidermal growth factor receptor 2, HER2/neu/erb-b2 1 Antimicrobial action sites, 325f
(17q21.1), 372–373 Antimer, 70
Kirsten rat sarcoma viral oncogene homolog, K-ras (12p 12), Antimicrobial agents, 324–329
375–376, 375f, 375t, 376f bacteriocidal, 324
leukemia and lymphoma molecular analysis bacteriostatic, 324
affinity maturation, 391b fungicidal, 324
Index 531
fungistatic, 324 Bacteriostatic, 324

mode of action, 325t Balanced chromosome, 187
molecular detection of resistance, 327–329 Balanced polymorphism, 181
resistance to, 302, 325–327 Balanced reciprocal translocation, 189f
Antimicrobial resistance in M. tuberculosis, 329 Band, 90–91, 99
Antiparallel, 9 Banding patterns, 396–397
Antisense strand, 59 Bar coding, 240, 241f
Apoptosis, 183, 183f, 371 Barr body, 345b
Apoptotic DNA, 183f Basal transcription complex, 28
APS. See Ammonium persulfate (APS) Base calling, 232
Apurinic site, 119f Base of RNA, 27
Arbitrarily primed PCR, 167–168, 169f, 332 Base pairing, 105
Array CGH, 363 Base pairs, 70
Array comparative genome hybridization technology, 363 Basic Local Alignment Search Tool (BLAST), 235t, 248
Array technology, 212–214 Basic PCR procedure, 143–147
bead array, 213 B-cell leukemia, 406t
high-density oligonucleotide arrays, 212 bDNA amplification, alkaline phosphatase, 172–173, 172f
redundant tiling, 212 Bead array technology, 138, 213, 213f
standard tiling, 212, 212f Bead arrays, serum antibody detection, 429f
Array-based hybridization, 133–138 Benzer, Seymour, 23, 44b
dot blot, 133–134 Bessman, Maurice J., 12b
genomic array technology, 134–138 Beta-lactam antibiotic resistance, 327–328
macroarrays in, 134 Beta-lactamase, reaction, 328f
microarrays in, 134–138, 135f Beta-pleated sheets, 42
microelectronic arrays, 135–138 Bilateral symmetry, 16
slot blot, 133–134 Bin, 273
Asexual reproduction, recombination in, 21–25 Binding sites, 8
conjugation, 21–23 Binning, 273, 273b
transduction, 23 Biochemical methods, 201–207
transformation, 23–25 alkaline phosphatase, 201, 202b, 202f
ASO. See Allele-specific oligomer hybridization (ASO) enzyme immunoassays, 201–202, 202b
ASR. See Analyte-specific reagents (ASR) gas chromatography, 204–205
Assign, 235t high-performance liquid chromatography, 204
Ataxia telangiectasia, 379 immunoassays, 201–202
Ataxia telangiectasia mutated gene, ATM (11q22), 379–380 immunosorbent assays, 202b
Attenuation, 63, 63f mass spectrometry, 205–207
Autologous bone marrow transplant, 283, 283f Biochemistry, nucleic acid, 2–37
Automated DNA isolation, 85f Biohazard stickers, 447f
Automated fluorescent sequencing, 229–235, 230f Bioinformatics, 248–250
cleaning the sequencing ladder, 232 algorithm, 249t
dye primer, 230, 231f alignment, 249t
dye terminator, 230, 231f annotation, 249t
electrophoresis, 231–232 computational biology, 248
sequence interpretation, 232, 233f–234f conservation, 249t
Autophagy, 349 domain, 249t
Autoradiograph, 469 gap, 249t
Autosomal STRs, 278 GenBank, 249t
Autosomal-dominant transmission, 347, 347f homology, 249t
Autosomal-recessive mutations, 348, 348f identity, 249t
Avuncular testing, 277–278 interface, 249t
local alignment, 249t
B motif, 249t
Bacteria, 309–313 multiple sequence alignment, 249t
DNA isolation, 80 optimal alignment, 249t
respiratory tract pathogens, 309–311 orthology, 249t
urogenital tract pathogens, 311–313 paralogy, 249t
Bacteriocidal, 324 PubMed, 249t
Bacteriocins, 26 query, 249t
Bacteriophage, 14, 23 in silico, 248
Bacteriophage T4, 24f similarity, 249t
532 Index
Bioinformatics (cont’d) Calibration verification, 457

SwissProt, 249t Calibrations, 456–457, 463
terminology, 249t Cambridge reference sequence, 291
Biology, molecular, 77–257 Cameras, gel photography, 462f
chromosomal structure and mutations, 179–198 Cameras and detectors, 462
DNA sequencing, 223–257 Cancer, defined, 370
gene mutations, 199–222 Cancer molecular basis, 371–372
nucleic acid amplification, 142–178 apoptosis, 371
nucleic acid and protein analysis and characterization, 112–141 cell division cycle, 371, 371f
nucleic acid extraction methods, 78–96 gain-of-function, 371
nucleic acid resolution and detection, 97–111 loss-of-function, 371–372
Biotin, 126, 127f oncogenes, 371
Bisulfite DNA sequencing, 236–237 tumor-suppressor genes, 371
BK/JC viruses, 323 Cap, 31, 32b
BLAST, 235t, 248 Capillary electrophoresis, 37, 102–104, 103f
Block thermal cyclers, 460, 461f Capillary gel electrophoresis, 269f, 384, 385f
Blocking, 123 Capillary transfer, 120, 120f
Blunt ends, 16 Capping, 31
Bone marrow aspirate. See Nucleated cells in suspension Carbon position numbering, nucleotide, 6f
Bone marrow engraftment testing, DNA polymorphisms, 283–289 Carboxy-terminal, 38
allogeneic transplant, 283 Carcinomas, 370
autologous transplant, 283 Cathode, 99, 120
chimera, 284 CDC assay example, 428t
fragment analysis, 286 CDC expression, 428t
full chimerism, 285 cDNA, 157, 165f
graft-versus-host disease, 284 CDR. See Complementarity determining region (CDR)
graft-versus-tumor, 284 Cell division cycle, 371, 371f
hematopoietic stem cells, 283 CEN probes, 192, 194f
informative locus, 285 Centrifuge speeds, 461f
linkage analysis, 282–283, 282f Centrifuges, 461
matched unrelated donor, 284 Centromere, 183, 184f
mixed chimerism, 285 Centromeric probe, 192, 194f
mosaic, 284b Certified chamber thermometers, 460f
myeloablative, 284, 287 CGH. See Comparative genome hybridization (CGH)
noninformative loci, 285 Chaperones, 49, 50f
post-transplant engraftment testing, 287–289, 287f Charging reaction, 46
pretransplant STR testing, 285–287 Chase, Martha, 23
split chimerism, 286b Chemical cleavage, 215–216
stutter, 286–287 Chemical safety, 465–466
Bordetella pertussis, 309 Chemical sequencing, 224–225, 224f
Bover, K., 418b Chimera, 284
Box-forkhead in rhabdomyosarcoma, 378 Chimerism, 345
Branched DNA amplification (bDNA), alkaline phosphatase, Chlamydia, 311
172–173, 172f Chlamydia trachomatis, 311
Break-apart probes, 192, 192f Chlamydophila pneumoniae, 311
Breast cancer 1 gene, BRCA1 (17q21), 380 Chromatin, 18, 65
Breast cancer 2 gene, BRCA2 (13q12), 380 Chromosomal abnormalities, 345–346
Brenner, Sydney, 43 aneuploidy, 345
Bridge PCR, 167, 168f. See also Surface amplification chimerism, 345
Buffer, agarose gels, 99 marker chromosomes, 346
Buffer systems, 104–106 mutations, 347t
additives, 105–106 polyploidy, 345
denaturing agents, 105 triploid, 346
ph drift, 106 Chromosomal mutations, 253t
conjugate, 104 Chromosomal structure, 181–186
Burkitt lymphoma, 406t aneuploid cells, 181
aneuploidy, 187f, 188f
C apoptotic DNA, 183f
C banding, 185b balanced polymorphism, 181
Calibration curve, 457 balanced reciprocal translocation, 189f
Index 533
break-apart probes, 192f framework regions, 393

centromere, 183, 184f hematological malignancies, gene mutations, 404–405
centromeric probe, 194f immunoglobulin heavy chain gene rearrangements, 391–394
chromosomal compaction, histones, 181–183, 182f immunoglobulin light chain gene rearrangements, 394
chromosome morphology, 183–184 mutations, hematological malignancies, 397–405
chromosome mutations, 181, 190f polyclonal lymphocytes, 391
classification by size and centromere position, 185t T-cell receptor gene rearrangements, 394–396
diploid, 180, 181 translocations, hematological malignancies
euploid, 181 RNA integrity control, 401
FISH analysis, 192f t(8;14)(q24;q11), 400, 400f
G-band patterns, 185f t(9;22)(q34;q11), 401–403, 401f, 402f, 403f
gene mutations, 181 t(11;14)(q13;q32), 398–399, 400f
genome, chromosomal mutations, 186–195 t(14;18)(q32;q21), 397–398, 397f, 398f, 399f
fluorescence in situ hybridization, 190–195 t(15;17)(q22;q11.2-q12), 403–404, 404f
karyotyping, 186–190 Clonotype, 396b
genome mutations, 181 Clustered, regularly interspaced, short palindromic repeat (CRISPR),
genotype, 180 17b
haploid, 180 CMV. See Cytomegalovirus (CMV)
human genome, 180 Coa typing, 335
karyotype, showing balanced reciprocal translocation, 189f Coagulase-negative Staphylococcus, 331f
male karyotype, 187f CODIS. See Combined DNA indexing system (CODIS)
metacentric chromosomes, 184f Codominant offspring, 347
metaphase chromosomes, 182f Codons, 45, 45f
mutation, 180–181 Coefficient of variance, 457, 461
phenotype, 180 Colicinogenic factors, 26
polymorphism, 180 Collection tubes
Robertsonian translocation, 189f for molecular testing, 448–450
sequence of nucleotides, 180 for nucleic acid testing, 449f, 449t
sizes of human chromosomes, 180t Comb, 106
staining pattern, chromosomes, 185f Combined DNA indexing system (CODIS), 272b
telomeric probes, 193f Combined paternity index (CPI), 276
trisomy, 181 Combined sibling index, 277
variant, 180 Combining typing results, 436, 436t
visualizing chromosomes, 184–186 Compaction, histones, 181–183, 182f
Chromosome, 43 Comparative genome hybridization (CGH), 136–137, 195, 195f, 196f
Chromosome mutations, 181, 190f Comparative genome hybridization technology, 196
Chromosome spread, 186 Comparative genomic arrays, 166
Chronic lymphocytic leukemia, 406t Complement, 44b
Chronic myelogenous leukemia, 406t Complementarity determining region (CDR), 393
Cis factors, 60, 61f Complementary DNA, 31, 157
Clades, 314 Complementary nucleotide, 7
Class switching, 388 Complement-dependent cytotoxicity, 427
Class-switch recombination, 389b Complete dominance, 347
Cleavage-based amplification, 173–174, 174f Complete penetrance, 349
Cleavase assay, 216, 218f Components of PCR, 144f, 144t, 147–151
Cleavase single-color assay, 218f Computational biology, 248
CLIA. See Clinical Laboratory Improvement Amendments (CLIA) Concatamers, 19
Clinical Laboratory Improvement Amendments (CLIA), 447, 470 Conformer, 207
Clinical sensitivity, 454, 454t Congenital diseases, 345
Clinical specificity, 454, 454t Conjugate, 104
Clonality Conjugated proteins, 43
detection. See Clonality detection Conjugating cells, 22f
molecular analysis of immunoglobulin heavy-chain gene clonality, Conjugation, 21–23
391–394, 392f, 393f, 394f Consensus, 42b
monoclonal, 391 Consensus primer, 393
polyclonal, 391 Consensus sequences, 248
Clonality detection, 391 Conservation, 249t
banding patterns, 396–397 Conservative, 201
complementarity determining regions, 393 Conservative substitutions, 200
consensus primer, 393 Constitutive RNA, 30, 60
534 Index
Contact precautions, 450 Deoxyribonuclease, 25

Contaminants, absorbance, 93t Deoxyribonuclease I, 18
Contamination, 150b Deoxyribonucleic acid. See DNA
Contamination control, 152–153, 305 Deoxyribonucleotide bases, 149–150
Continuous allele system, 266 Deoxyribose, 3
Controls Depurination, 118, 119f
PCR, 152–156 Derivative chromosome, 188
PCR contamination, 153–154 Detection
test performance, 457–458 microorganisms, 301–343
Coordination of HLA test methods, 437 AFLP analysis, 334f
Corepressor, 63 amplification controls, 306f
Corey, Robert, 42 antimicrobial action sites, 325f
C0 t value, 128 antimicrobial agents, 302, 324–329
C0 t½, 128 bacteria, 309–313
Coverage, 247 beta-lactamase, reaction, 328f
CpG islands, 66, 68f coagulase-negative Staphylococcus, 331f
CPI. See Combined paternity index (CPI) epidemiological typing methods, 330t
CREG. See Cross-reactive epitope group (CREG) fluorescent AFLP analysis, 335f
Crick, Francis, 43, 44b, 45 fungi, 323–324
CRISPR enzyme systems, 115–116, 116f genes conferring resistance to antimicrobial agents, 326t
crRNA, 115 genital tract organisms, 312t
Criteria for accepting specimens, 447–448, 448f genomes, human viruses, 314t
Crossing over, 20, 20f HIV viral loads, nucleic acid amplification methods, 318t
Crossmatching molecular detection, microorganism, 308–324
antibodies, 426, 427f molecular epidemiology, 329–337
serological analysis, 429, 430f nucleic acid amplification tests, viruses, 315t–317t
Cross-reactive epitope group (CREG), 428 nucleic acid test, target sequences, 307f
Crude DNA extraction, 86f parasites, 324
Cryostat, 202 penicillin, 328f
Cryotubes, 451, 453f penicillin structure, 328f
CT, 160 PFGE pattern interpretation, 332t
Curve analysis, homozygous mutant, 209f quality control, 305–306
Cut points, 458 RAPD gel results, 333f
Cut-off values, 458 resistance mechanisms, 326t
Cycle sequencing, 229 respiratory tract organisms, 310t
Cycles, 144 reverse transcriptase, 302
Cycling probe, 174 ribosomal RNA genes, 336f
Cycling probe fluorescence, 174f sample preparation, 304–305
Cystic fibrosis, 353–354, 354f sequence targets, 307
Cystic fibrosis transmembrane conductance, 353, 354f specimen collection, 302–304
Cytidine, 4 specimen transport systems, 303t
Cytochrome P-450 enzymes, 354–355, 354f, 355f Swab Extraction Tube System, 303f
Cytomegalovirus (CMV), 320–321 vancomycin structure, 329f
Cytopathic effect, 313–314 vancomycin-resistant S. aureus, 327f
Cytosine, 3 viral load measurement, test performance, 318t
Cytosine residues, 238f viruses, 313–323
Cytotoxicity, cells stained for, 428f nucleic acid, 97–111
agarobiose, 99f
D agarose, 99t
DAPI. See Diamidino-2-phenylindole (DAPI) buffer systems, 104–106
ddNTP. See Dideoxynucleotide (ddNTP) capillary electrophoresis, 103f
Deaza dGTP, 149b detection systems, 109–111
Deletion, 187 double-stranded DNA, 100f
Denaturation double-stranded DNA fragments, 102f
amplification program, 144 dye comigration, 109t
DNA, 119 electrophoresis, 98
DNA target, 145f electrophoresis equipment, 106–109
Denaturing agents, 8b, 105 field inversion gel electrophoresis, 100f
Denaturing gels, 123 gel loading, 108–109
Deoxynucleotide triphosphates (dNTP), 143, 146f gel systems, 99–102
Index 535
horizontal gel electrophoresis, 98f electrophoresis, 231–232

horizontal submarine gel, 106f sequence interpretation, 232, 233f–234f
peristaltic pump, 106f manual sequencing, 224–229
polyacrylamide, 101f chemical sequencing, 224–225, 224f
polyacrylamide concentration, 103t dideoxy chain termination (Sanger) sequencing, 225–229,
polyacrylamide electrophoresis combs, 107f 225f, 226f
vertical gel apparatus, 108f Maxam-Gilbert sequencing, 224–225
Detection limit, 454t, 455 Discrete allele systems, 268
Detection of clonality. See Clonality detection Discriminatory capacity, 279
Detection of gene mutations, 201–218 Discriminatory power, 337
biochemical methods, 201–207 Dispersive, 10b
chemical cleavage, 215–216 DNA, 2–20
Cleavase assay, 216, 218f adaptors, 16
enzymatic and chemical cleavage methods, 215–217 adenine, 3
heteroduplex analysis with single-strand specific nucleases, 216 adenosine, 4
hybridization-based methods antiparallel, 9
allele-specific oligomer hybridization, 209 bacteriocins, 26
array technology, 212–213 bacteriophage, 14, 23
heteroduplex analysis, 211–212 bacteriophage T4, 24f
melt-curve analysis, 209–211 bilateral symmetry, 16
single-strand conformation polymorphism, 207–209 blunt ends, 16
methods, 217–218 carbon position numbering, nucleotide, 6f
nonisotopic RNase cleavage assay, 216, 217f colicinogenic factors, 26
nucleic acid analyses, 207–218 complementary, 16
restriction fragment length polymorphisms, 215–216, 215f, 216f complementary nucleotide, 7
sequencing-based methods, 213–215 concatamers, 19
allelic discrimination with fluorogenic probes, conjugating cells, 22f
214–215, 215f CpG islands, 66
sequence-specific PCR, 213–214 crossing over, 20, 20f
Detection systems, 109–111, 129–132 cytidine, 4
fluorescent dyes, 109–110 cytosine, 3
microorganisms, genes conferring resistance to antimicrobial demethylation, 69
agents, 326t denaturing agents, 8b
nonradioactive detection, 130, 131f deoxyribonuclease, 25
nucleic acid-specific dyes, 110 deoxyribonuclease I, 18
probe labeled with radioactive phosphorous atoms, 130, 130f deoxyribose, 3
signal-to-noise ratio, 132 dispersive, 10b
silver stain, 110–111 DNA polymerase, 12f, 13f
dGMP. See Nucleotides deoxyguanosine monophosphate (dGMP) DNA replication, 8f
Diamidino-2-phenylindole (DAPI), 186 DNA synthesis, 9f
Dideoxy chain termination (Sanger) sequencing, 225–229 double helix, 4b, 5f, 7b, 9b
alkaline phosphatase, 227b double stranded, 8
ddNTP derivatives added, 226f double-strand break, 16, 19
dideoxynucleotide, 226, 227f endonucleases, 14
internal labeling, 226 enzymes metabolizing DNA, 14–19
M13, 226b episomal nature of the resistance factor, 25–26
M13 universal primer, 226b episomes, 26
manual, 225f exonuclease, 12
sequencing ladder, 228, 229f, 230 exonuclease I, 17
Dideoxynucleotide (ddNTP), 226, 226f, 227f exonuclease II, 18
Diffuse large B-cell lymphoma, 406t exonuclease III, 17
Digoxygenin, 126 exonuclease VII, 17
Dioxetane substrates, light emitted, 130f fertility factor, 21, 22f
Diploid, 180 fidelity, 14
Diploid organisms, 181 gamete, 14
Direct sequencing, 224–235 gap-filling DNA polymerase, 11b
automated fluorescent sequencing, 229–235, 230f guanine, 3
cleaning the sequencing ladder, 232 guanosine, 4
dye primer, 230, 231f gyrases, 19
dye terminator, 230, 231f hemimethylated, 19
536 Index
DNA (cont’d) replication complex, 11b

heterosis, 19 replication fork, 10
Hfr, 23 replisome, 11b
histone code, 65 restriction enzymes, 14–17, 15t, 16f
holoenzyme, 11–12 restriction modification, 7b
homologous sequences, 9f ribonuclease, 24–25
hybridization, 9 RNase H, 11b
hydrogen bonds, 7 S1 nuclease, 18
imprinting, 66 satellite plasmid, 26
intercalating agents, 8b semiconservative, 9
Klenow fragment, 12 sexually reproducing organisms, recombination in, 19–21
lagging strand, 10 simultaneous replication, 10f
leading strand, 10 single-strand break, 19
ligase, 13 sticky ends, 16
major groove, 8b structure, 4–9
meiosis, 20 substrate specificity, 14
Mendel, 19b–20b sugar-phosphate backbone, 8
methylation, 66–68 supercoiled, 19
micrococcal nuclease, 18 supercoiled plasmids, 26, 26f
migration, 25 template, 9
minor groove, 8b terminal transferase, 14
mismatches, 7 thymidine, 4
monomeric, 14 thymine, 3, 14b
mung bean nuclease, 18 topoisomerases, 19
mutations, 12 traits, 3
nick, 13 transduction, 23
nick translation, 13, 13f transformation, 23–25
nitrogen base, 3, 7 transforming factor, 24f, 25
nuclease BaI31, 18 DNA extraction/storage cards, 86
nuclein, 3 DNA fingerprinting, 265–266, 266b
nucleoside, 4, 5f DNA isolation, 79–87
nucleotide deoxyguanosine 5′ phosphate, 5f bacteria and fungi, 80
nucleotides, 3 inorganic isolation methods, 83
nucleotides deoxyguanosine monophosphate, 6f isolation of mitochondrial DNA, 86
Okazaki fragments, 10 lipoproteins, 81
oligonucleotides, 18 nucleated cells in suspension, 80
overhangs, 16 organic isolation methods, 81–83
palindromic, 16 plasma, 80–81
phosphodiester bond, 3 preparing sample, 79–81
plasmids, 23, 25–26, 26f proteolytic lysis, 85–86
polymerase classified by sequence homology, 11t salting out, 83
polymerases, sequence homology classification, 11t solid-phase isolation, 84–85, 84f
primase, 10 specimen sources, 79t
processivity, 14 tissue samples, 81
proofread, 13 viruses, 80
protease, 24–25 DNA metabolizing enzymes, 17–19
purines, 4 DNA polymerase, 12f, 13f, 146f, 150–151
pyrimidine dimers, 14b DNA polymorphisms, 260–300
pyrimidines, 4 allele peak locations, 269f
pyrophosphate exchange, 12 amelogenin locus, 270f
pyrophosphorolysis, 12 autologous bone marrow transplant, 283f
R factors, 26 bone marrow engraftment testing, 283–289
radioactive protein, 24f allogeneic transplant, 283
recBC nuclease, 18 autologous transplant, 283
recognition sites, 8b chimera, 284
recombinant, 19, 21 fragment analysis, 286
recombination, 19, 21f, 22f full chimerism, 285
recombination in asexual reproduction, 21–25 graft-versus-host disease, 284
recombined chromosomes, 22f graft-versus-tumor, 284
replication, 9–14 hematopoietic stem cells, 283
Index 537
informative locus, 285 DNA probes, 123–124

linkage analysis, 282–283, 282f DNA replication, 8f
matched unrelated donor, 284 DNA sequencing, 223–257
mixed chimerism, 285 Assign, 235t
mosaic, 284b bioinformatics, 248–250
myeloablative, 284, 287 algorithm, 249t
noninformative loci, 285 alignment, 249t
post-transplant engraftment testing, 287–289, 287f annotation, 249t
pretransplant STR testing, 285–287 computational biology, 248
split chimerism, 286b conservation, 249t
stutter, 286–287 domain, 249t
capillary gel electrophoresis, 269f gap, 249t
electropherogram, 276f, 278f GenBank, 249t
epigenetic profiles, 294 homology, 249t
HLA-DRB1 gene, 421f identity, 249t
linkage analysis, 282–283, 282f interface, 249t
microvariant allele, 272f local alignment, 249t
mitochondrial DNA polymorphisms, 291–293 motif, 249t
mitochondrial genome, 291f multiple sequence alignment, 249t
PAGE analysis, 285f optimal alignment, 249t
paternity tests, 276t orthology, 249t
polymorphisms, 262t paralogy, 249t
protein-based identification, 293–294 PubMed, 249t
quality assurance for surgical sections using STR, 289, 289f query, 249t
RFLP inheritance, 264f in silico, 248
RFLP typing, 262–266 similarity, 249t
alleles, 263 SwissProt, 249t
genetic mapping with RFLPs, 264 terminology, 249t
heterozygous, 263 bioinformatics terminology, 249t
homozygous, 263 bisulfite DNA sequencing, 236–237
human identification, RFLP, 265–266 BLAST, 235t
locus, 263 consensus sequences, 248
parentage testing, RFLP, 264–265, 265f cytosine residues, 238f
short tandem repeat TH01, 267f dideoxynucleotide, 226f
short tandem repeats, 268f direct sequencing, 224–235
single nucleotide polymorphisms, 289–291 automated fluorescent sequencing, 229–235, 230f
Human Haplotype Mapping project, 290–291, 290f, 291b manual sequencing, 224–229
Southern blot, 262t electropherogram, 233f
types, 290t Factura, 235t
STR locus information, 271t FASTA, 235t
STR typing by PCR, 266–278 fluorescent sequencing chemistries, 231f
allelic ladders, 268 GRAIL, 235t
discrete allele systems, 268 Human Genome Project, 250–254, 250t, 251t, 253t
gender identification, 270, 270f IUB universal nomenclature, 248
internal size standards, 268 Basic Alignment Search Tool, 248
microsatellites, 267 hierarchal shotgun sequencing, 251, 252f
microvariants, 267 for mixed bases, 250t
mini-STR, 268 manual dideoxy sequencing, 225f
short tandem repeats, 267 Matchmaker, 235t
STR analysis, 268–278 Maxam-Gilbert sequencing, 224t, 225f
STR nomenclature, 270 next-generation sequencing, 238–248, 239b, 239f,
test results analysis, 270, 272, 272t 456
tandem repeat, 265f Phrap, 235t
types of polymorphisms, 261 Phred, 235t
Y-STR, 278–282 Polyphred, 235t
autosomal STRs, 278 pyrogram, 236
genotypes, 281t pyrosequencing, 235–236, 236f
locus information, 280t–281t RNA sequencing, 237–238
matching with Y-STRs, 279, 281–282 SeqScape, 235t
paternal lineage test, 279 sequencing ladder, 230
538 Index
DNA sequencing (cont’d) Dye blobs, 232

software programs, sequence data, 235t Dye comigration, 109t
TIGR Assembler, 235t Dye primer, 230, 231f
DNA synthesis, 9f Dye terminator, 230, 231f
DNA template, 149
DNA-based tissue typing, 417–445, 418 E
additional recognition factors, 437–438 E site, 49
allografts, 418 EBNA. See EBV nuclear antigen (EBNA)
bead arrays, serum antibody detection, 429f EBV. See Epstein-Barr virus (EBV)
CDC assay example, 428t EBV nuclear antigen (EBNA), 320
CDC expression, 428t EGF. See Epidermal growth factor (EGF)
crossmatching, antibodies, 427f EGFR. See Epidermal growth factor receptor (EGFR)
cytotoxicity, cells stained for, 428f EIA. See Enzyme immunoassays (EIA)
DNA polymorphisms, 421f Electrodes, 99
graft-versus-host disease, 418 Electroendosmosis, 99
HLA polymorphisms, 420–425 Electropherogram, 232, 233f, 276f, 278f
allele, 420, 421–422 Electrophoresis, 231–232
haplotype, 420, 421f agarose, 98
HLA nomenclature, 420, 422–425 anode, 98
HLA type, 420 defined, 98
KIR and HLA gene interactions, 438t electropherogram, 232
KIR gene cluster, 438f measurement, 90–91
laboratory testing summary, 439–440 polyacrylamide, 98
major histocompatibility complex, 418, 419t Electrophoresis capillary replacement, 459f
MHC disease association, 438–439 Electrophoresis equipment, 106–109
MHC locus, 418–420 comb, 106
molecular analysis of MHC, 425–437 shark tooth combs, 107
combining typing results, 436, 436t submarine gels, 106
coordination of HLA test methods, 437 wells, 106
crossmatching, 427 Electrophoretic transfer, 120, 121f
DNA-based typing, 430–436 ELISA. See Immunosorbent assays (ELISA)
HLA test discrepancies, 436–437 Elongation, transcription, 28–29, 28f
serological analysis, 425–437 Emulsion PCR, 166–167
polypeptides, 420f End labeling, 126
reverse dot-blot SSOP, 431–432, 432f Endonucleases, 14
SSOP assay, 431f Endpoint analysis, 159
transplant evaluation, 439t Engraftment, 284
DNA-based typing, 430–436 Enhancers, 63
alignments of HLA polymorphic regions, 431 Enterobacterial repetitive intergenic consensus (ERIC), 333, 335f
next-generation sequencing libraries, 239f, 435f Enzymatic and chemical cleavage methods, 215–217
other methods, 435–436 Enzyme adaptation (induction), 61, 61b
sequence-based typing, 433–435, 433f, 434f Enzyme immunoassays (EIA), 201–202, 202b
sequence-specific oligonucleotide probe hybridization, 431–432 Enzyme induction, 63
sequence-specific PCR, 432–433, 432f, 433f Enzyme repression, 63
DNAse I, 18 Enzymes metabolizing DNA, 14–19
dNTP. See Deoxynucleotide triphosphates (dNTP) DNA metabolizing enzymes, 17–19
Documentation of test results, 468–469 helicases, 18–19
gene nomenclature, 469 ligase, 17
gene sequencing results, 469 methyltransferases, 19
reporting results, 469–470 nucleases, 17–18
resequencing, 469 restriction enzymes, 14–17, 15t
summary sheet or database, 470 Epidemic, 329
Domains, 38 Epidemiological typing methods, 330t
Dominant, 438 Epidermal growth factor (EGF), 374f
Dominant-negative phenotype, 347, 348f Epidermal growth factor receptor, EGFR (7p12), 373–375
Dot blot, 133–134, 134f, 209 Epigenetic factor classification, 69
Double helix, 4b, 5f, 7b, 9b Epigenetic profiles, 294
Double-strand break, 16, 19 Epigenetic regulation, 66
Double-stranded DNA, 8, 102f Epigenetics, defined, 65
Dual-fusion probes, 191 Episomal nature of the resistance factor, 25–26
Index 539
Episomes, 26 Fungi
Epitopes, 123, 202 detection of, 323–324
Epstein-Barr virus (EBV), 320 DNA isolation, 80
ER stress, 51b Fungicidal, 324
ERIC. See Enterobacterial repetitive intergenic consensus (ERIC) Fungistatic antimicrobial agents, 324
ESI spectrophotometry, 205–206, 206f Fusion gene, 401
Ethidium bromide, 90, 91f FXS. See Fragile X syndrome (FXS)
Euchromatin, 67b, 183
Eukaryote(s), 29, 30f, 58t, 63f G
Euploid organisms, 181 G1 checkpoint, 371
E-value, 248 G2 checkpoint, 371
Ewing sarcoma, EWS (22q12), 376–377, 377t, 378f Gain-of-function mutations, 347, 371
Exclusion, 265, 272, 276 Gamete, 14
Exons, 32 Gamow, George, 44, 44b
Exonuclease II, 18 Gap, 249t, 252
Exonucleases, 12, 17 Gap-filling DNA polymerase, 11b
Expect value, 248 Gas chromatography, 204–205, 205f
Explosive materials, 450f, 465f G-banding, 185, 185b, 185f, 186f
Expression arrays, 136 Gel, 99
Extended haplotype, 281b Gel electrophoresis, 155f, 461f
Extended MHC locus (xMHC), 418 Gel loading, 108–109
Extension/termination assay, 146b Gel mobility shift assay, 139, 139f
Extraction with chelating resin, 86 Gel photography, cameras, 462f
Gel systems, 99–102
F agarose gels, 99–101
Factor V Leiden mutation, 349, 352, 352f buffers, 99
Factura, 235t capillary electrophoresis, 102–104
False negative, 153 field-inversion gel electrophoresis, 100
False positive, 306 polyacrylamide gels, 101–102
FASTA, 235t pulsed-field gel electrophoresis, 100–101
Fertility factor, 21, 22f GenBank, 248, 249t
F factor, 23 Gender identification, 270, 270f
Fidelity, 14, 150 Gene, 43, 43f
Field inversion gel electrophoresis (FIGE), 100, 100f Gene expression, 27, 58
FIGE. See Field inversion gel electrophoresis (FIGE) Gene mutations, 181, 199–222
FISH analysis, 192f allele-specific primer amplification, 214f
Flammable materials, 465f allelic discrimination, 215f
Flemming, Walther, 3 antigen detection, 202f
Fluorescence, TaqMan signal, 162f bead array technology, 213f
Fluorescence in situ hybridization, 190–195 cleavase assay, 216, 218f
interphase FISH, 190–195, 191f cleavase single-color assay, 218f
metaphase FISH, 193–194 curve analysis, homozygous mutant, 209f
Fluorescent AFLP analysis, 335f detection of gene mutations, 201–218
Fluorescent dyes, 109–110 biochemical methods, 201–207
Fluorescent resonance energy transfer, 162–163, 163f, 210–211, chemical cleavage, 215–216
210f, 211f enzymatic and chemical cleavage methods, 215–217
Fluorescent sequencing chemistries, 231f heteroduplex analysis with single-strand specific
Fluorometry, 93–94 nucleases, 216
Follicular lymphoma, 406t methods, 217–218
FR. See Framework region (FR) nonisotopic RNase cleavage assay, 216, 217f
Fragile X chromosome, 359f nucleic acid analyses, 207–218
Fragile X syndrome (FXS), 358–360, 359f, 360f restriction fragment length polymorphisms, 215–216,
Fragment analysis, 286 215f, 216f
Frameshift mutation, 200, 201b, 219 sequencing-based methods, 213–215
Framework region (FR), 393 ESI spectrophotometry, 205–206, 206f
FRET. See Fluorescent resonance energy transfer), 162–163, 163f, frameshift mutation, 200, 201b, 219
210–211, 210f, 211f gas chromatography, 205f
FRET probes, 163f gene names, 219–220
Full chimerism, 285 gene variant nomenclature, 218–219
Fume hoods and laminar flow hoods, 462–463 heteroduplex analysis, 212f
540 Index
Gene mutations (cont’d) metalloproteins, 43

liquid chromatography, 205f monomer, 43
MALDI spectrophotometry, 205–206, 206f nonpolar, 38
MALDI-TOF spectrophotometry, 206, 206f nonsense codons, 45
melt-curve analysis, 209f nonsense-mediated decay, 51
multiplex allele-specific PCR, 214f oligomers, 43
multiplex PCR, 216, 216f P site, 47, 49
mutation detection methodologies, 208t peptide, 38
NIRCA analysis, 217f peptide bond, 38, 41f
PCR-RFLP, 215–216, 215f peptidyl transferase, 49
point mutations, 200, 200t polar, 38
sequence-specific primer amplification, 213f polypeptides, 38
single-strand conformation polymorphism, 208f primary structure, 41
types of, 200–201 prions, 43
Gene names, 219–220 prosthetic group, 43
Gene nomenclature, 469 protein synthesis, 50f
Gene panels, 240 proteins, 38, 43–44, 45f
Gene rearrangements, 387–397 proteome, 38
banding patterns, 396–397 proximal regulatory sequences, 43
defined, 387 pyrrolysine, 38b
detection of clonality, 391 quaternary structure, 43
immunoglobulin heavy-chain gene rearrangement in B cells, random coils, 42
387–389, 388f regulatory sequences, 43
immunoglobulin light-chain, 394, 395f release factors, 51
immunoglobulin light-chain gene rearrangement in B cells, ribosomal binding site, 47
389–390, 389f ribosome(s), 47, 48f
molecular analysis of immunoglobulin heavy-chain gene clonality, RNA Tie Club, 44b
391–394, 392f, 393f, 394f secondary structure, 42
T-cell receptor, 390–391, 390f, 390t, 391f, 394–396, 395f, 396f selenocysteine, 38b
V(D)J recombination, 387, 387f selenoproteins, 38b
Gene sequencing results, 469 side chain, 38
Gene variant nomenclature, 218–219 structural sequences, 43
Gene/chromosomal mutations, solid tumors, 372–387 suppressor tRNAs, 47b
Genes conferring resistance to antimicrobial agents, 326t tertiary structure, 42–43
Genetic code, 37–46 translation, 46–51
alpha helix, protein, 42, 42f amino acid charging, 46–47
amino acid(s), 38–43 protein synthesis, 47–51, 50f
biosynthetic groups of, 40t transmembrane proteins, 40f
structures, 39f tRNA charging, 46
amino acyl tRNA synthetases, 47f zinc finger, 42b
amino terminal, 38 zwitterions, 38
aminoacyl tRNA synthetases, 46 Genetic concordance, 272
anticodon, 45–47 Genetic mapping with RFLPs, 264
beta-pleated sheets, 42 Genetic profiling, 265–266, 266b
carboxy terminal, 38 Genital tract organisms, 312t
chaperones, 49 Genome, chromosomal mutations, 186–195
chromosome, 43 fluorescence in situ hybridization, 190–195
codons, 45, 45f interphase FISH, 190–195, 191f
complement, 44b metaphase FISH, 193–194
conjugated proteins, 43 karyotyping, 186–190
consensus, 42b balanced translocation, 187
domains, 38 chromosome spread, 186
E site, 49 defined, 186
ER stress, 51b deletion, 187
extracellular domain, 38 derivative chromosome, 188
gene, 43 descriptive abbreviations, 191t
glycoproteins, 43 insertion, 187–188
isodecoders, 46 inversions, 188
leucine zipper, 42b isochromosome, 188, 189f
lipoproteins, 43 microdeletions, 187
Index 541
mitogen, 186 Hazard labels, 466f

paracentric inversions, 188 HCV. See Hepatitis C virus (HCV)
pericentric inversions, 188 HeLa cervical carcinoma, 33t
reciprocal translocations, 186 Helicases, 11b, 18–19, 37
ring chromosome, 188 Hemachromatosis, 352–353, 353f, 354f
robertsonian, 187 Hematological malignancies
translocations, 186 gene mutations, 404–405
unbalanced translocation, 187 CCAAT/enhancer-binding protein, alpha (CEBPA), 404
Genome mutations, 181, 346t FMS-related tyrosine kinase 3, 404–405
Genome sequencing, 12b Janus kinase 2, 405
Genomes, human viruses, 314t nucleophosmin/nucleoplasmin family, member 1 (NPM1), 404
Genomic amplification methods, 165–168 molecular analysis, 387–406
arbitrarily primed PCR, 167–168, 169f molecular targets
bridge PCR, 167, 168f kappa deleting element, 389
comparative genomic arrays, 166 T-cell receptor gene rearrangement, 390–391
emulsion PCR, 166–167 trimming, 391
multiple displacement amplification, 166 mutations
randomly amplified polymorphic DNA, 167 lymphoid, 397–400
solid-phase emulsion PCR, 166–167, 167f myeloid, 401–405
surface amplification, 167, 168f Hematopoietic stem cells, 283
whole-genome amplification, 165, 165f, 166, 166f Hemimethylated, 19
Genomic array technology, 134–138 Hemizygous for X-linked genes, 348
macroarrays in, 134 Hepatitis C virus, 321–322
microarrays in, 134–138, 135f Herpes simplex virus (HSV), 320
microelectronic arrays, 135–138 Herpes virus (VZV), 320–322
Genomic DNA fragments, 118f cytomegalovirus, 320–321
Genomic imprinting, 345, 362–363 Epstein-Barr virus, 320
Genomics, 136b herpes simplex virus, 320
Genotype, 180 Hershey, Alfred, 23
Germline, 387–388 Heterochromatin, 67b, 183
Gill, Peter, 275b Heteroduplex analysis, 211, 212f, 216
Gloves, 450f Heteroduplexes, 98
Glycopeptide antibiotic resistance, 328–329 Heterologous extrinsic controls, 305
Glycoproteins, 43 Heterologous intrinsic controls, 305
Gonadal mosaicism of new mutations, 348b, 355–356, 356f Heteronuclear RNA, 30f, 32
Gorer, P., 418b Heterophagy, 349
G-proteins, 376 Heteroplasmy, 293
Graft failure, 285 Heterosis, 19
Graft-versus-host disease (GVHD), 284, 418 Heterozygosity loss, 385, 386f
Graft-versus-tumor (GVT), 284 Heterozygous, 263
GRAIL, 235t HFE protein, 353f
GTPase-activating proteins, 376 Hfr, 23
Guanine, 3 Hierarchal shotgun sequencing, 251, 252f
Guanosine, 4 High-density oligonucleotide arrays, 136, 212
GVHD. See Graft-versus-host disease (GVHD) Highly active antiretroviral therapy (HAART), 317
GVT. See Graft-versus-tumor (GVT) High-performance liquid chromatography (HPLC), 204
Gyrases, 19 High-positive controls, 457
High-resolution banding, 186
H Hirschsprung disease, 381
HAART. See Highly active antiretroviral therapy (HAART) Histocompatibility antigens, 437
Hairpin, 29, 59 Histone(s), 181–183
Hairy cell leukemia, 406t code of, 67b, 67t, 68f
Haploid genes, 180 modification of, 65–66, 66f, 68f
Haplotype, 278, 420, 421f HIV. See Human immunodeficiency virus (HIV)
Haplotype diversity, 279 HLA. See Human leukocyte antigens (HLA)
HapMap project, 146b, 253, 290–291, 290f, 291b Hoagland, amino acid charging studies, 46
Haptens, 125 Hodgkin disease, 370
Hardy-Weinberg equilibrium, 274b Hoechst 33258, 94
Harvey rat sarcoma viral oncogene homolog, H-ras (11p15), Holding and storage requirements, 451
375–376, 375f, 375t Holley, Robert, 35b
542 Index
Holocentric chromosomes, 184b short tandem repeat TH01, 267f, 268f

Holoenzyme, 11–12, 35f single nucleotide polymorphisms, 289–291, 290t
Homoduplexes, 211 Southern blot, 262t
Homogeneous MassExtend, 146b STR locus information, 271t
Homologous, 66 STR typing by PCR, 266–278
Homologous extrinsic controls, 305 allelic ladders, 268
Homologous partners, 207–208 discrete allele systems, 268
Homologous sequences, 9f gender identification, 270, 270f
Homology, 248, 249t internal size standards, 268
Homozygous, 263 microsatellites, 267
Horizontal gel electrophoresis, 98f microvariants, 267
Horizontal submarine gel, 106f mini-STR, 268
Hot-start PCR, 154–155 short tandem repeats, 267
HPLC. See High-performance liquid chromatography (HPLC) STR analysis, 268–278
HPV. See Human papillomavirus (HPV) STR nomenclature, 270
HSV. See Herpes simplex virus (HSV) test results analysis, 270, 272, 272b, 272t, 273b
HUGO. See Human Genome Organization (HUGO) tandem repeat, 265f
Human epidermal growth factor receptor 2, HER2/neu/erb-b2 1 types of polymorphisms, 261
(17q21.1), 372–373 Y-STR, 278–282
Human genome, 180 autosomal STRs, 278
Human Genome Organization (HUGO), 219, 270b genotypes, 281t
Human Genome Project, 250–254, 251t locus information, 280t–281t
Human Haplotype Mapping Project, 146b, 253, 290–291, matching with Y-STRs, 279, 281–282
290f, 291b paternal lineage test, 279
Human identification, 260–300 Human immunodeficiency virus (HIV), 314, 317–319
allele peak locations, 269f genotyping, 319
amelogenin locus, 270f HIV genotyping, 319
autologous bone marrow transplant, 283f limit of detection, 317–318
bone marrow engraftment testing, DNA polymorphisms, 283–289 primary resistance mutations, 319
allogeneic transplant, 283 secondary resistance mutations, 319
autologous transplant, 283 viral load, 317, 318t
chimera, 284 Human leukocyte antigens (HLA)
fragment analysis, 286 alleles identified serologically and by DNA sequence, 426t
full chimerism, 285 classes, 418, 419f
graft-versus-host disease, 284 defined, 418
graft-versus-tumor, 284 HLA and KIR gene interactions, 438t
hematopoietic stem cells, 283 nomenclature, 420, 422–425
informative locus, 285 ambiguity, 425
linkage analysis, 282–283, 282f null allele, 425
matched unrelated donor, 284 resolution of allele detail, 425
mixed chimerism, 285 split specificities, 422
mosaic, 284b synonymous changes, 425
myeloablative, 284, 287 polymorphisms, 420–425
noninformative loci, 285 allele, 420, 421–422
post-transplant engraftment testing, 287–289, 287f haplotype, 420, 421f
pretransplant STR testing, 285–287 type, 420
split chimerism, 286b serologically defined specifications, 422t–424t
stutter, 286–287 test discrepancies, 436–437
capillary gel electrophoresis, 269f type, 420
electropherogram, 276f, 278f typing, 427–428
epigenetic profiles, 294 Human papillomavirus (HPV), 322
linkage analysis, 282–283, 282f Humoral sensitization, 428
microvariant allele, 272f Hungerford, David, 188b
mitochondrial DNA polymorphisms, 291–293, 291f Huntingtin repeat expansion, 361f
PAGE analysis, 285f Huntington disease, 361
paternity tests, 276t Hybrid, 157
polymorphisms, 262t Hybrid capture assays, 173, 173f
protein-based identification, 293–294 Hybrid resistance, 437
quality assurance for surgical sections using STR, 289, 289f Hybrid vigor, 14
RFLP in, 262–266, 264f Hybridization, 9
Index 543
Hybridization conditions, stringency, 128–129 resistance mechanisms, 326t

C0 t value, 128 respiratory tract organisms, 310t
C0 t½, 128 reverse transcriptase, 302
melting temperature, 128, 128f ribosomal RNA genes, 336f
sequence complexity, 128 sample preparation, 304–305
Hybridization technologies, 116–121, 117t sequence targets, 307
northern blot, 122 specimen collection, 302–304
Southern blot, 117–121 specimen transport systems, 303t
western blot, 122–123 Swab Extraction Tube System, 303f
Hybridization-based methods vancomycin structure, 329f
allele-specific oligomer hybridization, 209 vancomycin-resistant S. aureus, 327f
array technology, 212–213 viruses, 313–323
heteroduplex analysis, 211 BK/JC viruses, 323
melt-curve analysis, 209–211 cytopathic effect, 313–314
single-strand conformation polymorphism, 207–209 hepatitis C virus, 321–322
Hybridomas, 126 herpes viruses, 320–321
Hydrogen bonds, 7 human immunodeficiency virus, 314, 317–319
Hypervariable region I, 291, 292f human papillomavirus, 322
Hypervariable region II, 291, 292f load measurement, test performance, 318t
mass spectrometry, 323
I mycology, 323–324
Iatrogenic, 330 nucleic acid amplification tests, 315t–317t
Identification, microorganisms, 301–343 respiratory viruses, 322–323
AFLP analysis, 334f Identity, 249t
amplification controls, 306f Idiopathic congenital central hypoventilation syndrome, 361–362
antimicrobial action sites, 325f Immobilized, 85
antimicrobial agents, 324–329 Immunoassays, 201–202
bacteriocidal, 324 Immunoglobulin, 389f
bacteriostatic, 324 Immunoglobulin heavy chain
fungicidal, 324 rearrangement of, 388f, 391–394, 392f, 393f
fungistatic, 324 rearrangement of B cells, 387–389, 388f
mode of action, 325t Immunoglobulin light chain gene rearrangement
molecular detection of resistance, 327–329 B cells, 389–390, 389f
resistance to, 302, 325–327 clonality detection, 394
bacteria, 309–313 kappa locus, 395f
respiratory tract pathogens, 309–311 lambda locus, 395f
urogenital tract pathogens, 311–313 Immunohistochemistry, 201, 202–204, 203f
beta-lactamase, reaction, 328f Immunosorbent assays (ELISA), 202b
coagulase-negative Staphylococcus, 331f Imprinting, 66
epidemiological typing methods, 330t Imprinting, genomic, 362–363
fluorescent AFLP analysis, 335f In silico, 248
fungi, 323–324 In vitro analytical test (IVAT), 465
genital tract organisms, 312t In vitro diagnostic (IVD), 464–465
genomes, human viruses, 314t Inclusion, 265
HIV viral loads, nucleic acid amplification methods, 318t IncRNA, 71–72, 73f
microorganisms, genes conferring resistance to antimicrobial Indexing, 240, 241f
agents, 326t Indirect nonradioactive detection, 130f
molecular detection, microorganism, 308–324 Inducible transcription, 30
molecular epidemiology, 329–337 Induction (enzyme adaptation), 61, 61b
epidemic, 329 Informative locus, 285
molecular strain typing, 330–336 Informative STR alleles, 286f
pandemic, 329 Inheritance of alleles, 282, 283f
typing method comparison, 336–337, 337t Inherited disease detection, 344–368
nucleic acid amplification tests, viruses, 315t–317t autosomal-dominant transmission, 347f
nucleic acid test, target sequences, 307f autosomal-recessive mutations, 348f
parasites, 324 chromosomal abnormalities, 345–346
penicillin structure, 328f chromosomal mutations, 253t
PFGE pattern interpretation, 332t cystic fibrosis, 353–354, 354f
quality control, 305–306 cystic fibrosis transmembrane conductance, 353, 354f
RAPD gel results, 333f cytochrome P-450 enzymes, 354–355, 355f
544 Index
Inherited disease detection (cont’d) Introns, 32

factor V Leiden mutation, 349, 352, 352f Invader assay, 174f
fragile X chromosome, 359f Inversions, 188
fragile X syndrome, 359f Investigational use only (IUO), 465
genome mutations, 346t Isochromosome, 188, 189f
genomic imprinting, 362–363 Isocratic, 204
gonadal mosaicism, 356f Isodecoders, 46
hemachromatosis, 352–353, 353f, 354f Isolation of DNA, 79–87
HFE protein, 353f bacteria and fungi, 80
Huntingtin repeat expansion, 361f inorganic isolation methods, 83
limitations to molecular testing, 363 isolation of mitochondrial DNA, 86
lysosome, 351f lipoproteins, 81
methylenetetrahydrofolate reductase enzyme, 352, 353f mitochondrial DNA, 86
mitochondrial deletion, 358f nucleated cells in suspension, 80
mitochondrial disorders, 357t organic isolation methods, 81–83
mitochondrial genome, 356f plasma, 80–81
mitochondrial mutations, 355f, 356–357 preparing sample, 79–81
molecular basis, inherited diseases, 345, 345b proteolytic lysis, 85–86
multimeric proteins, 347, 348f salting out, 83
NARP mitochondrial point mutation, 358f solid-phase isolation, 84–85, 84f
nonpolyglutamine expansion disorders, 363t tissue samples, 81
pedigree, 347f viruses, 80
prothrombin, 349–350, 351t, 352 Isolation of RNA, 87–90
sequence-specific PCR, 352f diethyl pyrocarbonate, 87b
single-gene disorders, 355–363 extraction of total RNA, 87
inheritance patterns, 346–349 intracellular, 88
molecular basis, 349–355 microfuge, 88–89
molecular methods, 350t polyA RNA, 89–90
nonclassical patterns, 355–363 proteolytic lysis of fixed material, 89
storage diseases, 351t RNAse-free, 87
thrombosis, 351t specimen collection, 87
triplet-repeat expansion, 359f, 361, 362f specimen sources, 90t
uniparental disomy, 362–363 total RNA, 87
X-linked recessive diseases, 348f Isotype, 388b
Initiation, transcription, 28 ITS. See Internal transcribed spacer (ITS)
Ink-jet technology, 135f IUB universal nomenclature, 248–250, 250t, 251, 252f, 253t
Inorganic DNA isolation, 83, 83f IUO. See Investigational use only (IUO)
Insertion, 187–188 IVAT. See In vitro analytical test (IVAT)
Instrument maintenance, 459–463 IVD. See In vitro diagnostic (IVD)
block thermal cyclers, 460
calibration verification, 457, 463 J
calibrations, 456–457, 463 Jacob, Francois, 23
cameras and detectors, 462 Janus kinase 2, 405
centrifuges, 461 Jeffrey, Alec, 275b
coefficient of variance, 457, 461 Joining reaction, 17b–18b
fume hoods and laminar flow hoods, 462–463
microcentrifuge speed controls, 461 K
power supplies, 461 Kammerer, Paul, 72b–73b
refrigerators and freezers, 459–460 Kappa deleting element (KDE), 389
reportable range, 455, 463 Karyotyping, 183, 186–190
Intercalating agents, 8b, 109–110 balanced chromosome, 187
Interface, 239, 249t chromosome spread, 186
Internal controls, 305, 457 defined, 186
Internal labeling, 226 deletion, 187
Internal size standards, 268 derivative chromosome, 188
Internal transcribed spacer (ITS), 310, 334 descriptive abbreviations, 191t
Interphase FISH, 190–195, 191f insertion, 187–188
Interpretation of results, 132–133 inversions, 188
Interspersed repetitive elements, 333–334 isochromosome, 188, 189f
Intervening sequences, 31, 31b microdeletions, 187
Index 545
mitogen, 186 Linkage disequilibrium, 282

paracentric inversions, 188 Linkage equilibrium, 274
pericentric inversions, 188 Linker DNA, 181, 183f
reciprocal translocations, 186 Lipoproteins, 43, 81
ring chromosome, 188 Liquid biopsy, 81, 386–387
robertsonian, 187 Liquid chromatography, 205f
showing balanced reciprocal translocation, 189f Local alignment, 249t
translocations, 186 Locked nucleic acids, 123
unbalanced translocation, 187 Locus, 263
KDE. See Kappa deleting element (KDE) Locus genotype, 272
Khorana, H. Gobind, 17b, 43, 45 Locus-specific brackets, 273b
Killer cell immunoglobulin-like receptors, 437–438 Locus-specific RFLP, 332
Kinetochore, 183, 184f Long interspersed nucleotide sequences (LINEs), 261
King, Mary Claire, 264b Long noncoding, 71–72
Kinship index, 277 Loss of heterozygosity (LOH), 385, 386f
KIR and HLA gene interactions, 438t Loss-of-function, 347, 371–372
KIR gene cluster, 438f Low-copy-number analysis, 275b
Kirsten rat sarcoma viral oncogene homolog, K-ras (12p 12), Low-positive controls, 457
375–376, 375f, 375t, 376f Lymphocytic leukemia, 406t
Klenow fragment, 12 Lymphoid malignancy mutations, 397–400
Kornberg, Arthur, 12b Lymphoma
Kornberg, Sylvy, 12b ALK proto-oncogene 2p23.1, 382
chromosomal abnormalities, 406t
L defined, 370
Labeling, probes, 126 gene rearrangements, 387–397
Labeling of reagents, 466–468 molecular analysis, 388–391
Laboratory techniques, 259–472. See also Molecular laboratory non-Hodgkin, 370
quality control Lynch syndrome, 382
DNA polymorphisms and human identification, 260–300 Lyon hypothesis, 345b
DNA-based tissue typing, 417–445 Lysosomal storage diseases, 349
inherited disease molecular detection, 344–368 Lysosome, 349, 351f
microorganism detection and identification, 301–343
molecular oncology, 369–416 M
quality assurance and quality control, 446–472 M13 universal primer, 226b
testing summary, 439–440 Macroarrays, 134
Laboratory-developed tests, 451 Major breakpoint region, 397
Lac operon, 62f Major groove, 8b
Lagging strand, 10 Major histocompatibility complex (MHC)
Laminar flow hoods, 462f disease association, 438–439
Leading strand, 10 genes, 419t
Leder, Philip, 43, 44 MHC locus, 418–420
Lederberg, Joshua, 23b extended MHC locus, 418
Legionella pneumophila, 309 mixed lymphocyte culture, 418
Lehman, I. Robert, 12b nonconventional antigens, 437
Leucine zipper, 42b MALDI spectrophotometry, 205–206, 206f
Leukemia MALDI-TOF spectrophotometry, 206, 206f
characteristic, 370 Male karyotype, 187f
chromosomal abnormalities, 406t Mantle cell lymphoma, 406t
gene rearrangements, 387–397 Manual dideoxy sequencing, 225f
molecular analysis, 388–391 Manual sequencing, 224–229
Leukocyte receptor cluster, 438 chemical sequencing, 224–225
Library, 240 dideoxy chain termination (Sanger) sequencing, 225–229
Ligase, 13, 17 alkaline phosphatase, 227b
Ligase chain reaction, 168–169, 169f dideoxynucleotide, 226, 227f
Likelihood ratio, 274 direct sequencing, 225f, 226f
Limit of detection, 305, 317–318 internal labeling, 226
Limitations to molecular testing, 363 M13 universal primer, 226b
Linearity, 454t sequencing ladder, 228, 229f, 230
LINEs. See Long interspersed nucleotide sequences (LINES) Maxam-Gilbert sequencing, 224–225
Linkage analysis, 282–283, 282f Marker chromosomes, 346
546 Index
Mass spectrometry, 205–207, 323, 336 Micrococcal nuclease, 18

MassExtend, 146b Microdeletions, 187
Master mix, 152 Microelectronic arrays, 134
Match probability, 273 Microfluidics, 94
Matched unrelated donor (MUD), 284 Microorganism detection, 301–343
Matching of profiles, 273–275 AFLP analysis, 334f
Matching with Y-STRs, 281–282 amplification controls, 306f
discriminatory capacity, 279 antimicrobial action sites, 325f
haplotype diversity, 279 antimicrobial agents, 302, 324–329
surname test, 282 bacteriocidal, 324
Matchmaker, 235t bacteriostatic, 324
Maxam-Gilbert sequencing, 224–225, 224t, 225f. See also Chemical fungicidal, 324
sequencing fungistatic, 324
Matrix Assisted Laser Desorption/Ionization – Time of Flight mode of action, 325t
(MALDI-TOF) spectrophotometry, 206, 206f molecular detection of resistance, 327–329
MCA. See Melt-curve analysis (MCA) resistance to, 302, 325–327
Measurement bacteria, 309–313
analyte measurement range, 454t respiratory tract pathogens, 309–311
electrophoresis, 90–91 urogenital tract pathogens, 311–313
fluorometry, 93–94 beta-lactamase, reaction, 328f
microfluidics, 94 coagulase-negative Staphylococcus, 331f
nucleic acid quality and quantity, 90–94 epidemiological typing methods, 330t
spectrophotometry, 92–93 fluorescent AFLP analysis, 335f
viral load measurement, test performance, 318t fungi, 323–324
mecA (methicillin resistance), 174 genital tract organisms, 312t
Meiosis, 20 genomes, human viruses, 314t
Melt-curve analysis (MCA), 209–211 HIV viral loads, nucleic acid amplification methods, 318t
fluorescent resonance energy transfer, 210–211, 210f, 211f microorganisms, genes conferring resistance to antimicrobial
PCR products, 209f agents, 326t
Melting temperature, 128, 128f molecular detection, microorganism, 308–324
Membrane types, 119–120 molecular epidemiology, 329–337
anode, 120 epidemic, 329
cathode, 120 molecular strain typing, 330–336
electrophoretic transfer, 120, 120f pandemic, 329
prehybridization, 121–122 typing method comparison, 336–337, 337t
vacuum transfer, 121 nucleic acid amplification tests, viruses, 315t–317t
MEN. See Multiple endocrine neoplasia (MEN) syndromes, 381 nucleic acid test, target sequences, 307f
Messenger RNA (mRNA), 29–33 parasites, 324
function, 27, 58 penicillin, 328f
processing, 31 penicillin structure, 328f
splicing, 31–33, 32f PFGE pattern interpretation, 332t
Metabolizing enzymes, RNA, 36–37 quality control, 305–306
Metacentric chromosome, 184, 184f RAPD gel results, 333f
Metalloproteins, 43 resistance mechanisms, 326t
Metaphase chromosomes, 182f, 194f respiratory tract organisms, 310t
Metaphase FISH, 193–194 reverse transcriptase, 302
spectral karyotyping, 193 ribosomal RNA genes, 336f
whole chromosome paints, 193, 194f sample preparation, 304–305
Metastasis, 370 sequence targets, 307
Methicillin and oxacillin, 328, 328f specimen collection, 302–304
Methicillin resistance (mecA), 174 specimen transport systems, 303t
Methicillin-resistant S. Aureus, 326 Swab Extraction Tube System, 303f
Methods of gene mutation detection, 217–218 vancomycin structure, 329f
Methylenetetrahydrofolate reductase, 352, 353f vancomycin-resistant S. aureus, 327f
Methyltransferases, 19 viral load measurement, test performance, 318t
mHag. See Minor histocompatibility antigens (mHag) viruses, 313–323
MHC. See Major histocompatibility complex (MHC) BK/JC viruses, 323
MIC. See Minimum inhibitory concentration (MIC) cytopathic effect, 313–314
Microarray(s), 134–138, 135f DNA isolation, 80
Microcentrifuge speed controls, 461 DNA template, 149
Index 547
hepatitis C virus, 321–322 crossmatching, 427

herpes viruses, 320–321 DNA-based typing, 430–436
human immunodeficiency virus, 314, 317–319 HLA test discrepancies, 436–437
human papillomavirus, 322 serological analysis, 425–437
mass spectrometry, 323 somatic hypermutation, 388
mycology, 323–324 Molecular basis, inherited diseases, 345, 345b
nucleic acid amplification tests, 315t–317t Molecular Beacons, 161–162, 162f
respiratory viruses, 322–323 Molecular biology, 77–257
MicroRNA (mRNA), 70 chromosomal structure and mutations, 179–198
Microsatellites DNA sequencing, 223–257
instability of, 382, 384–385, 385f gene mutations, 199–222
STR typing by PCR, 267 nucleic acid amplification, 142–178
Microtome, 202 nucleic acid and protein analysis and characterization, 112–141
Microvariant allele, 272f nucleic acid extraction methods, 78–96
Microvariants, 267 nucleic acid resolution and detection, 97–111
Miescher, Johann Friedrich, 3, 3b–4b Molecular detection, microorganism, 308–324
Migration of DNA, 25 Molecular detection of resistance, 327–329
Minimal haplotype, 281b antimicrobial resistance in M. tuberculosis, 329
Minimum inhibitory concentration (MIC), 327 beta-lactam antibiotic resistance, 327–328
Minisatellites, 267 glycopeptide antibiotic resistance, 328–329
Mini-STR, 268 methicillin, 328
Minor cluster region, 397 minimum inhibitory concentration, 327
Minor groove, 8b susceptibility testing, 327
Minor groove-binding dyes, 110 Molecular epidemiology, 329–337
Minor histocompatibility antigens (mHag), 437 epidemic, 329
miRNA. See MicroRNA (mRNA) molecular strain typing, 330–336
miRNA silencing, 72f pandemic, 329
Mismatch repair (MMR), 382, 383t, 384f typing method comparison, 336–337, 337t
Mismatches, 7 Molecular laboratory quality control, 305–307, 446–472. See also
Mispriming, 148, 148f Laboratory techniques
Mitochondrial deletion, 356, 358f acrylic shielding, 466, 467f
Mitochondrial disorders, 357t amplification control, 305, 457
Mitochondrial DNA polymorphisms, 291–293 biohazard stickers, 447f
Anderson reference, 291 block thermal cyclers, 461f
Cambridge reference sequence, 291 cameras, gel photography, 462f
hypervariable region I, 291, 292f centrifuge speeds, 461f
hypervariable region II, 291, 292f certified chamber thermometers, 460f
Oxford sequence, 291 Clinical Laboratory Improvement Amendments, 447, 470
Mitochondrial genome, 291f, 356f collection tubes, nucleic acid testing, 449f, 449t
Mitochondrial mutations, 355f, 356–357 contamination controls, 305
Mitogen, 186 cryotubes, 451, 453f
Mitosis, 181 documentation, test results, 468–469
Mixed chimerism, 285 electrophoresis capillary replacement, 459f
Mixed leukocyte reaction, 429–430 explosive materials, 450f, 465f
Mixed lymphocyte culture (MLC), 418 flammable materials, 465f
MLC. See Mixed lymphocyte culture (MLC) gel electrophoresis, 461f
MLST. See Multilocus sequence typing (MLST) gloves for handling explosive materials, 450f
MMR. See Mismatch repair (MMR) hazard labels, 466f
Mobility, 139 heterologous extrinsic, 305
Modified bases, 7b heterologous intrinsic, 305
Molecular abnormalities, 382 homologous extrinsic, 305
Molecular analysis instrument maintenance, 459–463
affinity maturation, 391b internal controls, 305
immunoglobulin light-chain gene rearrangement in B cells, laminar flow hoods, 462f
389–390 negative quality control, 457
immunoglobulins heavy-chain gene clonality, 391–394, 392f, negative template control, 305
393f, 394f nucleic acid storage, 453t
MHC, 425–437 positive controls, 305, 457
combining typing results, 436, 436t radionuclides, 467t
coordination of HLA test methods, 437 reagent blank, 305
548 Index
Molecular laboratory quality control (cont’d) polycythemia vera, 406t

reagents, 463–468 pre-B acute lymphoblastic leukemia, 406t
sensitivity control, 305, 457 proto-oncogenes, 381
specimen handling, 447–451 rearranged during transfection (RET) proto-oncogene
specimen storage, 452t (10q11), 381
test performance, 451, 453–458 sequencing panels, 405
test performance measurements, 454t synovial sarcoma translocation, chromosome 18-synovial
true negative, 305 sarcoma breakpoint 1 and 2, SYT-SSX1, SYT-SSX2 t(X;18)
validation, 306 (p11.2;q11.2), 377–378
Molecular laboratory testing summary, 439–440 T-cell lymphoma, 406t
Molecular oncology, 369–416 T-chronic lymphocytic leukemia, 406t
analytical targets, molecular testing, 372 tissue-specific, 372
acute lymphocytic leukemia, 406t tumor protein 53, TP53 (17p13), 378–379
acute monocytic leukemia, 406t tumor-specific, 372
acute myeloid leukemia, 406t V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene
acute myeloid leukemia/myelodysplastic syndrome, 406t homolog, Kit, c-KIT (4q12), 382
acute myelomonocytic leukemia, 406t V-myc avian myelocytomatosis viral-related oncogene,
acute nonlymphocytic leukemia, 406t neuroblastoma-derived, 381
acute promyelocytic leukemia, 406t Von Hippel-Lindau gene, VHL (3p26), 380–381
acute T-lymphocytic leukemia, 406t V-Ros Avian UR2 Sarcoma Virus Oncogene Homolog 1
anaplastic lymphoma receptor tyrosine kinase (ALK) proto- (ROS1) Proto-Oncogene (6q22.1), 381
oncogene, 2p23.1, 382 Waldenström macroglobinemia, 406t
aneuploidy, 372 cancer molecular basis, 371–372
ataxia telangiectasia mutated gene, ATM (11q22), 379–380 capillary gel electrophoresis, 385f
B-cell leukemia, 406t cell division cycle, 371f
breast cancer 1 gene, BRCA1 (17q21), and breast cancer 2 epidermal growth factor, 374f
gene, BRCA2 (13q12), 380 EWS translocation partners, 377t
Burkitt lymphoma, 406t gene rearrangements. See Gene rearrangements
chronic lymphocytic leukemia, 406t germline, 387–388
chronic myelogenous leukemia, 406t immunoglobulin, 389f
clonality detection, 391–405 immunoglobulin heavy-chain gene, 388f, 391–394, 392f, 393f
diffuse large B-cell lymphoma, 406t immunoglobulin heavy-chain gene rearrangement, B cells,
epidermal growth factor receptor, EGFR (7p12), 373–375 387–389, 388f
Ewing sarcoma, EWS (22q12), 376–377, 377t, 378f immunoglobulin light-chain kappa locus, 395f
follicular lymphoma, 406t immunoglobulin light-chain lambda locus, 395f
gene/chromosomal mutations, solid tumors, 372–387 leukemias, chromosomal abnormalities, 406t
hairy cell leukemia, 406t microsatellite instability, 385f
Harvey rat sarcoma viral oncogene homolog, H-ras (11p15), mismatch repair system, 383t, 384f
375–376, 375f, 375t neoplasm classification, 370–371
hematological malignancies, molecular analysis, 387–406 p53 mutation analysis, 379t
hematological malignancies, molecular targets, 387–397 ras proteins, 377f
heterozygosity loss, 385 receptor tyrosine kinases, 373f
human epidermal growth factor receptor 2, HER2/neu/erb-b2 RT-PCR, 378f
1 (17q21.1), 372–373 solid tumors, molecular abnormalities, 383t
Kirsten rat sarcoma viral oncogene homolog, K-ras (12p 12), T-cell receptor gene, 390f, 390t, 395f, 396f
375–376, 375f, 375t, 376f V(D)J recombination, 387, 387f
leukemia and lymphoma molecular analysis, 388–391 Molecular strain typing, 330–336
leukemias, chromosomal abnormalities, 406t amplified fragment length polymorphism, 332–333
liquid biopsy, 386–387 arbitrarily primed PCR, 332
lymphomas, chromosomal abnormalities, 406t coa typing, 335
mantle cell lymphoma, 406t enterobacterial repetitive intergenic consensus, 333, 335f
microsatellite instability, 382, 384–385, 385f internal transcribed spacer elements, 334
molecular abnormalities, 382 interspersed repetitive elements, 333–334
mucosa-associated lymphoid tissue lymphoma, 406t ITS1, 334
multiple myeloma, 406t ITS2, 334
myeloproliferative/myeloblastic disease, 406t locus-specific RFLP, 332
myeloproliferative/myelodysplastic disease, 406t mass spectrometry, 336
neuroblastoma ras, N-ras (1p13), 375–376, 375f, 375t multilocus sequence typing, 336, 336t
paired box-forkhead in rhabdomyosarcoma, PAX3-FKHR, PCR-RFLP, 332
PAX7-FKHR, t(1;13), t(2;13), 378 plasmid analysis, 331
Index 549
pulsed-field gel electrophoresis, 331 National DNA Database (NDNAD), 275

REP-PCR, 333–334 National DNA Index System (NDIS), 275
restriction fragment length polymorphism, 331–332 NDIS. See National DNA Index System (NDIS)
ribotyping, 332 NDNAD. See National DNA Database (NDNAD)
spa typing, 334–335 Negative controls, 457
Molecular-based detection methods, 310t, 312t Negative template control, 153, 305
Monoclonal antibodies, 126 Neisseria gonorrhoeae, 311
Monoclonal (monotypic) clonality, 391 Neoplasm, defined, 370
Monomer, 43 Neoplasm classification, 370–371
Monomeric, 14 Nested PCR, 157–158, 158f
Morphology, chromosome, 183–184 Neuroblastoma ras, N-ras (1p13), 375–376, 375f, 375t
Mosaic, 284b New mutations, 348b
Motif, 249t Next-generation sequencing, 238–248
mRNA. See Messenger RNA (mRNA) filtering and annotation, 247, 247t
MRSA. See Methicillin-resistant S. Aureus (MRSA) gene panels, 240
MSI. See Microsatellite instability (MSI) Human Genome Project, 239b
Mucosa-associated lymphoid tissue lymphoma, 406t library, 239f, 435f
MUD. See Matched unrelated donor (MUD) library preparation, 239f, 240–241, 241f
Mullis, Kary, 143, 145b, 146b sequence quality, 246
Multicolor FISH, 194, 194f sequencing platforms, 243–246, 244f, 245f, 246f
Multidrug-resistant N. gonorrhoeae, 326 targeted libraries, 241–242, 242f, 243f
Multifactorial inheritance, 363 test performance, 456
Multilocus sequence typing (MLST), 336, 336t Nick, 13
Multimeric proteins, 347, 348f Nick translation, 13, 13f, 126
Multiple displacement amplification, 166 NIRCA. See Nonisotopic RNase cleavage assay (NIRCA)
Multiple endocrine neoplasia (MEN) syndromes, 381 NIRCA analysis, 217f
Multiple locus probe, 265–266 Nirenberg, Marshall, 43, 44
Multiple myeloma, 406t Nitrocellulose, 117
Multiple sequence alignment, 249t Nitrogen bases, 3, 4
Multiplex allele-specific PCR, 214f NMD. See Nonsense-mediated decay (NMD)
Multiplex PCR, 156, 216, 216f, 286f Noncoding RNAs, 69–73
Mung bean nuclease, 18 Nonconservative substitution, 200
Mutation Nonconventional MHC antigens, 437
chromosomal, 181, 186–195, 186f–195f Nondisjunction, 346
copying errors resulting in, 12 Nonhistone proteins, 181, 183
defined, 180 Non-Hodgkin lymphoma, 370
detection methodologies, 208t Noninformative loci, 285
gain-of-function, 347 Nonisotopic RNase cleavage assay (NIRCA), 216, 217f
gene, 181, 199–222. See also Gene mutations Nonpolar, 38
genome, 181 Nonpolyglutamine expansion disorders, 363t
gonadal mosaicism, 348b Nonradioactive detection, 132t
hematological malignancies, 397–405 Nonsense codons, 45
lymphoid malignancies, 397–400 Nonsense substitution, 200
in mitochondrial genes, 356–357 Nonsense-mediated decay (NMD), 51
myeloid malignancy, 401–405 Non-sequence-specific dyes, 160f
nuclear gene mutation disorders, 358t NOR. See Nucleolar organizing region (NOR) staining
spectra, 405 Northern blot, 122, 133f
Mycobacterium tuberculosis, 309–310 Nosocomial, 330
Mycology, 323–324 Nowell, Peter, 188b
Mycoplasma pneumoniae, 310–311 NTPs, 150
Mycoplasma spp., 313 Nuclear gene mutation disorders, 358t
Myeloablative, 284, 287 Nuclease BaI31, 18
Myeloid malignancy mutations, 401–405 Nucleases, 17–18
Myeloproliferative/myeloblastic disease, 406t Nucleated cells in suspension, 80
Myeloproliferative/myelodysplastic disease, 406t Nucleic acid, 8–9, 112–141
amplification, tests of, 315t–317t
N amplification of, 142–178
Nanney, David, 65 analyses, 207–218
NARP mitochondrial point mutation, 358f apurinic site, 119f
NASBA. See Nucleic acid sequence-based amplification (NASBA) biochemistry of, 2–37
550 Index
Nucleic acid (cont’d) capillary gel electrophoresis, 385f

biotin, side chain, 127f cell division cycle, 371f
capillary transfer, 120, 120f defined, 370
characterization, 112–141 epidermal growth factor, 374f
detection of, 97–111 EWS translocation partners, 377t
dioxetane substrates, light emitted, 130f gene rearrangements. See Gene rearrangements
dot blot, 134f immunoglobulin, 389f
electrophoretic transfer, 120, 121f immunoglobulin heavy-chain gene, 388f, 392f, 393f
extraction of, 78–96 immunoglobulin light-chain kappa locus, 395f
gel mobility shift assay, 139f immunoglobulin light-chain lambda locus, 395f
genomic DNA fragments, 118f leukemias, chromosomal abnormalities, 406t
hybridization technologies, 116–121, 117t microsatellite instability, 385f
indirect nonradioactive detection, 130f mismatch repair system, 383t, 384f
nonradioactive detection, 132t neoplasm classification, 370–371
northern blot, 133f p53 mutation analysis, 379t
peptide nucleic acids, 123, 125f ras proteins, 377f
phosphodiester, nucleic acids, 125f receptor tyrosine kinases, 373f
photolithographic target synthesis, 136f RT-PCR, 378f
probe design of, 126–128 solid tumors, molecular abnormalities, 383t
probe types of, 124 T-cell receptor gene, 390f, 390t, 395f, 396f
resolution of, 97–111 Oncomirs, 70
restriction enzyme mapping, 113–115, 114f, 115f 1000 Genomes Project, 253–254
sample labeling, 137f Open reading frame, 31
sequence-based amplification of, 164–165 Operator, 61
single-stranded DNA, reannealing, 129f Operon, 61, 61f
solution hybridization, 138f Optimal alignment, 249t
Southern blot, 133f Organic DNA isolation, 82f
storage of, 453t Organic isolation methods, 81–83
test, target sequences, 307f Organic RNA extraction, 88, 88f
testing collection tubes, 449f, 449t Orthology, 249t
vacuum transfer, 121, 121f Overhangs, 16
Nucleic acid sequence-based amplification (NASBA), 164–165 Oxacillin, 328f
Nucleic acid-specific dyes, 110 Oxford sequence, 291
Nuclein, 3
Nucleolar organizing region (NOR) staining, 186 P
Nucleolus, 28, 58 P site, 47, 49
Nucleoside, 4, 5f p53 mutation analysis, 379t
Nucleosome, 65, 181 PAGE. See Polyacrylamide gel electrophoresis (PAGE)
Nucleotide deoxyguanosine 5′ phosphate, 5f PAGE analysis, 285f
Nucleotide repeat expansion disorders, 357–362 Paired box-forkhead in rhabdomyosarcoma, PAX3-FKHR,
fragile X syndrome, 358–360, 359f, 360f PAX7-FKHR, t(1;13), t(2;13), 378
Huntington disease, 361 Palindromic, 16
idiopathic congenital central hypoventilation syndrome, 361–362 PAM. See Protospacer adjacent motif (PAM)
trinucleotide repeat, 362 Pandemic, 329
triplet-repeat expansion, 361, 362f Panel reactive antibodies (PRA), 429
Nucleotide triphosphate, 10, 126 Paracentric inversions, 188
Nucleotides, 3, 4–8 Paralogy, 249t
Nucleotides deoxyguanosine monophosphate (dGMP), 6f Parasites, 324
Null allele, 425 Parentage testing, RFLP, 264–265, 265f
Partial dominance, 347
O Paternity index, 275–276
Okazaki fragments, 10 Paternity tests, 276t
Oligo polythymine columns, 90f Pauling, Linus, 42
Oligoclone, 391b PCR. See Polymerase chain reaction (PCR)
Oligomers, 43, 165f Pedigree, 346, 347f
Oligonucleotides, 18 Penetrance, 349
Oncogenes, 371 Penicillin structure, 328f
Oncology, 369–416 Peptide, 38
analytical targets, molecular testing, 372 Peptide bond, 38, 41f
cancer molecular basis, 371–372 Peptide nucleic acids (PNAs), 123, 125f
Index 551
Peptidyl transferase, 49 DNA polymerase, 150–151

Pericentric inversions, 188 DNA template, 149
Peristaltic pump, 106f elements, 144t
PFGE. See Pulsed-field gel electrophoresis (PFGE) endpoint analysis, 159
PFGE pattern interpretation, 332t fidelity, 150
ph drift, 106 fluorescent resonance energy transfer, 162–163, 163f
Phasing of alleles, 434 Homogeneous MassExtend, 146b
Phenotype, 180 hot-start, 154–155
Phosphodiester bond, 3, 125f Human Haplotype Mapping Project, 146b
Phosphor, 134 hybrid, 157
Photobleaching, 193 mass spectrometry of viruses, 323
Photographic equipment, 462 master mix, 152
Photolithographic target synthesis, 136f mispriming, 148, 148f
Phrap, 235t modifications, 156–163
Phred, 235t Molecular Beacons, 161–162, 162f
Plasma, DNA isolation, 80–81 multiplex, 156
Plasma cell, 370 negative template control, 153
Plasmid analysis, 331 nested PCR, 157–158, 158f
Plasmids, 23, 25–26, 26f NTPs, 150
PNAs. See Peptide nucleic acids (PNAs) PCR-RFLP, 215–216, 215f, 332
Point mutations, 200, 200t prevention of mispriming, 154
Polar, 38 primer dimers, 148, 149f
Polarity, 8 primers, 147–150, 148b
Polonies, 243 product cleanup, 155, 156f
PolyA polymerase, 36 psoralens, 153
PolyA tail, 31, 31b pyrimidine dimers, 154b
Polyacrylamide quantitative, 158–164, 159f
electrophoresis of nucleic acids, 98 quencher, 162
repeating unit acrylamide, 101f rapid, 152
Polyacrylamide concentration, 103t reaction, 152, 152f
Polyacrylamide electrophoresis combs, 107f reagent blank, 152
Polyacrylamide gel electrophoresis (PAGE), 101–102 real-time, 152, 158–164, 159f
Polyadenylate polymerase, 31, 59 reverse transcriptase, 150, 157
Polyadenylation, 31 scorpion, 162, 163f
Polyadenylation signal, 29 semi-nested, 157–158
Polyadenylic acid, 31 sequence-specific, 156–157
Polycistronic RNA, 29 spin columns, 155, 156f
Polyclonal antibodies, 124–125 Stoffel fragment, 150
Polyclonal (polytypic) clonality, 391 strand, 143
Polyclonal lymphocytes, 391 SYBR green, 160
Polycythemia vera, 406t tailed primers, 149, 149f
Polymerase, 28f, 58t Taq polymerase, 150
Polymerase chain reaction (PCR), 143–164 TaqMan, 160, 161f
amplicons, 143, 147f template, 143
amplification control, 153 thermal cyclers, 151–152
amplification program, 144 threshold cycle, 160
annealing, 145, 146f, 147b touchdown, 155
basic PCR procedure, 143–147 Tth polymerase, 150
buffer, 151 viruses, 149
complementary DNA, 31, 157 Polymerase classified by sequence homology, 11t
components, 144f, 144t, 147–151 Polymerases, 11–14, 35–36
contamination, 150b Polymorphisms, 180, 262t
contamination control in, 153–154 Polypeptides, 38, 420f
control, 152–156 Polyphred, 235t
CT, 160 Polyploidy, 345
cycles, 144 Polythymine, 31
deaza dGTP, 149–150 Position effect, 67b, 181
denaturation, 144 Positive controls, 305, 457
deoxynucleotide triphosphates, 143, 146f Post-PCR, 153
deoxyribonucleotide bases, 149b Power supplies, 461
552 Index
PRA. See Panel reactive antibodies (PRA) Protease, 24–25

Preanalytical error, 447 Protein analysis, 112–141
Pre-B acute lymphoblastic leukemia, 406t apurinic site, 119f
Pre-PCR, 153 array-based hybridization, 133–138
Precautions in sample processing, 450 biotin, side chain, 127f
Precision, 454t capillary transfer, 120, 120f
Prehybridization, 121–122 detection systems, 129–132
Premutations, 359 nonradioactive detection, 130, 131f
Preparation of resolved DNA for blotting, 118–119 probe labeled with radioactive phosphorous atoms, 130, 130f
Preparing the DNA sample, 79–81 signal-to-noise ratio, 132
Prevention of mispriming, 154 dioxetane substrates, light emitted, 130f
Primary antibody, 123 dot blot, 134f
Primary resistance mutations, 319 electrophoretic transfer, 120, 121f
Primary structure, 41 gel mobility shift assay, 139f
Primase, 10 genomic DNA fragments, 118f
Primer dimers, 148, 149f hybridization conditions, stringency, 128–129
Primer PCR. See Sequence-specific PCR (SSP-PCR) hybridization technologies, 116–121, 117t
Primers northern blot, 122
DNA replication, 11b Southern blot, 117–121
nucleic acid amplification, 147–150, 148b, 148f, 149f western blot, 123–124
reagents, 463–464, 463f indirect nonradioactive detection, 130f
TaqMan probes, 161f ink-jet technology, 135f
Prions, 43 interpretation of results, 132–133
Prior odds, 276–277, 277t nonradioactive detection, 132t
Private antibodies, 428 northern blot, 133f
Probability of paternity, 276–277 peptide nucleic acids, 123, 125f
Proband, 385 phosphodiester, nucleic acids, 125f
Probe, 117 photolithographic target synthesis, 136f
Probe amplification, 168–171 probes, 123–128
ligase chain reaction, 168–169, 169f restriction enzyme mapping, 113–115, 114f, 115f
Qβ replicase, 170–171, 171f sample labeling, 137f
strand displacement amplification, 169–170, 170f, 171f single-stranded DNA, reannealing, 129f
Probes, 123–128 solution hybridization, 138–139, 138f
DNA probes, 123–124 Southern blot, 133f
locked nucleic acids, 123 vacuum transfer, 121, 121f
nucleic acid probe design, 126–128 western blot, 122–123, 123b, 127b, 127f, 133f
nucleic acid probe types, 124 Protein gel electrophoresis, 430
peptide nucleic acids, 123, 125f Protein probes, 124–126
probe labeling, 126 alternative nucleic acids, 126b
protein probes, 124–126 haptens, 125
RNA probes, 124 hybridomas, 126
secondary antibody, 123 monoclonal antibodies, 126
Probit analysis, 455 polyclonal antibodies, 124–125
Processivity, 14 Protein synthesis, 47–51, 50f
Product rule, 274 Protein truncation tests, 380
Proficiency testing, 468 Protein-based identification, 293–294
Profile, human identification, 266 Proteins, 37–46
Profile matching, 273–275 A site, 49, 49f
Hardy-Weinberg equilibrium, 274b alpha helix, 42, 42f
likelihood ratio, 274 amino acids, 38–43, 39f, 40t
linkage disequilibrium, 282 amino acyl tRNA synthetases, 47f
linkage equilibrium, 274 amino terminal, 38
match probability, 273 aminoacyl tRNA synthetases, 46
product rule, 274 anticodon, 45–47
subpopulations, 274 beta-pleated sheets, 42
Prokaryotes, 30f, 62f, 63f carboxy terminal, 38
Prokaryotic RNA polymerase, 35f chaperones, 49
Promoter, 28, 58 chromosome, 43
Proofread, 13 codons, 45, 45f
Prosthetic group, 43 complement, 44b
Index 553
conjugated proteins, 43 Prothrombin, 349–350, 351t, 352

consensus, 42b Proto-oncogenes, 381
defined, 37 Protospacer, 17b
domains, 38 Protospacer adjacent motif (PAM), 17b
E site, 49 Proximal regulatory sequences, 43
ER stress, 51b Pseudogenes, 261
extracellular domain, 38 Psoralens, 153
gene, 43, 43f Public antigens, 428
genetic code, 38, 43–44, 45f PubMed, 249t
glycoproteins, 43 Pulsed-field gel electrophoresis (PFGE), 100–101, 331, 332t
isodecoders, 46 Purines, 4
leucine zipper, 42b Pyrimidine dimers, 14b, 154b
lipoproteins, 43 Pyrimidines, 4
metalloproteins, 43 Pyrogram, 236
monomer, 43 Pyrophosphate, 32b
nonpolar, 38 Pyrophosphate exchange, 12
nonsense codons, 45 Pyrophosphorolysis, 12
nonsense-mediated decay, 51 Pyrosequencing, 235–236
oligomers, 43 defined, 236f
P site, 47, 49 pyrogram, 236
peptide, 38 Pyrrolysine, 38b
peptide bond, 38, 41f
peptidyl transferase, 49 Q
polar, 38 Q banding, 184–185, 185f
polypeptides, 38 Qβ replicase, 170–171, 171f
primary structure, 41 Quality assurance
prions, 43 molecular laboratory quality control, 458–459
prosthetic group, 43 surgical sections using STR, 289
protein synthesis, 50f testing, 289f
proteome, 38 Quality control. See Molecular laboratory quality control
proximal regulatory sequences, 43 Quantitative FISH, 195b, 195f
pyrrolysine, 38b Quantitative PCR, 158–164, 159f
quaternary structure, 43 Quaternary structure, 43
random coils, 42 Quencher, 160, 162
regulatory sequences, 43 Query, 248, 249t
release factors, 51
ribosomal binding site, 47 R
ribosomes, 47, 48f R banding, 185b
RNA Tie Club, 44b R factors, 26
secondary structure, 42 Radioactive chemicals, 465, 467f
selenocysteine, 46b Radioactive protein, 24f
selenoproteins, 46b Radionuclides, 467t
side chain, 38 Random coils, 42
structural sequences, 43 Random priming, 126
suppressor tRNAs, 47b Randomly amplified polymorphic DNA, 167
tertiary structure, 42–43 RAPD gel results, 333f
translation, 46–51 Rapid extraction methods, 86
amino acid charging, 46–47 Rapid lysis methods, 86
protein synthesis, 47–51, 50f Rapid PCR, 152
transmembrane proteins, 40f Ras proteins, 377f
tRNA charging, 46 Read alignment, 246
zinc finger, 42b Reagent alcohol, 468
zwitterions, 38 Reagent blank, 152
Proteolytic lysis Reagent blank controls, 305
DNA extraction/storage cards, 86 Reagents, 463–468
extraction with chelating resin, 86 analyte-specific reagents, 464
of fixed material, 85–86 categories, 464–465
rapid extraction methods, 86 chemical safety, 465–466
rapid lysis methods, 86 investigational use only, 465
Proteome, 38, 136b labeling, 466–468
554 Index
Reagents (cont’d) capillary electrophoresis, 103f

proficiency testing, 468 double-stranded DNA, 100f
reference range, 465 double-stranded DNA fragments, 102f
research use only, 465 dye comigration, 109t
storage, 466 field inversion gel electrophoresis, 100f
in vitro analytical test, 465 horizontal gel electrophoresis, 98f
in vitro diagnostic, 464–465 horizontal submarine gel, 106f
Real-time PCR, 152, 158–164, 159f peristaltic pump, 106f
Reannealing, single-stranded DNA, 129f polyacrylamide, 101f
Rearranged during transfection proto-oncogene (10q11), 381 polyacrylamide concentration, 103t
recBC nuclease, 18 polyacrylamide electrophoresis combs, 107f
Receptor tyrosine kinases, 372, 373f vertical gel apparatus, 108f
Recessive, 438 Respiratory tract pathogens, 309–311
Reciprocal translocations, 186 Bordetella pertussis, 309
Recognition sites, 8b Chlamydophila pneumoniae, 311
Recombinant DNA, 19, 21 Legionella pneumophila, 309
Recombination, 22f Mycobacterium tuberculosis, 309–310
Recombination activating genes, 388 Mycoplasma pneumoniae, 310–311
Recombination DNA, 19, 21f, 22f Streptococcus pneumoniae, 311
Recombination in asexual reproduction, 21–25 typical organisms, 310t
conjugation, 21–23 Respiratory viruses, 322–323
transduction, 23 Restriction endonuclease fingerprinting (REF-SSCP), 209b
transformation, 23–25 Restriction enzyme cutting and resolution, 117–118
Recombination in sexually reproducing organisms, 19–21 Restriction enzyme mapping, 113–115
Recombined chromosomes, 22f Restriction enzymes, 14–17, 15t, 16f
Redundant tilting, 212 Restriction fragment length polymorphism (RFLP)
Reference range of tests, 454t, 465 BamH1, 215f
Refrigerators and freezers, 459–460 defined, 115
REF-SSCP. See Restriction endonuclease fingerprinting (REF-SSCP) inheritance, 264f
Regulation of transcription, 60–65 molecular epidemiology, 331–332
epigenetics, 65–69 multiplex PCR, 216f
regulation of RNA synthesis at initiation, 60–64 nucleic acid analyses, 215–216
Regulatory sequences, 43 single-nucleotide polymorphisms, 261
Release factors, 51 typing, 262–266
Repairing polymerases, 14b alleles, 263
Replication, 9–14 genetic mapping with RFLPs, 264
Replication complex, 11b heterozygous, 263
Replication factor (RF), 226 homozygous, 263
Replication fork, 10 human identification, RFLP, 265–266
Replisome, 11b locus, 263
Reportable range of tests, 454t, 455, 463 parentage testing, RFLP, 264–265, 265f
Reporter dye, 174 Restriction map, 113
Reporting results, 469–470 Restriction mapping, 114f, 115f
REP-PCR, 333–334 Restriction modification, 7b
Repressor, 61 Reverse dot blot, 134, 209
Reproducibility of test results, 454t Reverse dot-blot SSOP, 431–432, 432f
Reproducible typing method, 337 Reverse transcriptase, 150
Research use only (RUO), 465 Reverse transcriptase PCR, 150, 157, 302
Resequencing, 469 Reverse transcription, 27b
Resistance mechanisms, 326t RF. See Replication factor (RF)
Resistance to antimicrobial agents, 325–327 RFLP. See Restriction fragment length polymorphism (RFLP)
multidrug-resistant N. gonorrhoeae, 326 Rhabdomyosarcoma, 378
multidrug-resistant S. aureus, 326 rho, 29
transposon, 327 Ribonucleases, 24–25, 36–37
Resolution Ribonucleic acid. See RNA
allele detail, 425 Ribose, 27
HLA typing methods, 436t Ribosomal binding site, 47
nucleic acid, 97–111 Ribosomal RNA (rRNA), 29, 30f, 336f
agarobiose, 99f Ribosomes, 33, 47, 48f
agarose, 99t Ribotyping, 332
Index 555
Ring chromosome, 188 regulation of transcription, 60–65

RISC. See RNA-induced silencing complex (RISC) repressor, 61
RNA, 27–37 reverse transcription, 27b
activator, 63 rho, 29
alpha phosphate, 29, 59 ribose, 27
alternative splicing, 33 ribosomal RNA, 29, 30f, 336f
anticodon, 34 ribosomes, 33, 47, 48f
antisense strand, 59 RNA interference, 70
attenuation, 63, 63f RNA-induced silencing complex, 71
base, 27 RNA-metabolizing enzymes, 36–37
base pairs, 70 sense strand, 59
cap, 31, 32b silencers, 63
chaperones, 49, 50f Sm proteins, 34b
chromatin, 18, 65 splicesosome, 34b
cis factors, 60, 61f splicing, 31–33
constitutive, 30, 60 trans factors, 60
corepressor, 63 transcription, 27–29
enhancers, 63 transfer RNA, 33–34, 35b
enzyme adaptation (induction), 61, 61b types/structures of RNA, 29–35
enzyme induction, 63 messenger RNA, 29–33
enzyme repression, 63 micro RNAs, 70
epigenetic regulation, 66 ribosomal RNA, 29, 30f
exons, 32 small interfering RNA, 70
gene expression, 27, 58 small nuclear RNA, 33, 33t
hairpin, 29, 59 small RNAs, 70–71
histone, 65–66 transfer RNA, 33–34, 35b
IncRNA, 71–72, 73f uracil, 27
inducible transcription, 30 RNA editing, 36b
intervening sequences, 31b RNA integrity control, 401
introns, 32 RNA interference (RNAi), 70
isolation, 87–90 RNA isolation, specimen sources, 90t
diethyl pyrocarbonate, 87b RNA polymerases, 27, 28t, 35–36, 35b, 58, 58t
extraction of total RNA, 87 RNA probes, 124
intracellular, 88 RNA sequencing, 237–238
microfuge, 88–89 RNA Tie Club, 44b
polyA RNA, 89–90 RNA-dependent RNA polymerases, 35
proteolytic lysis of fixed material, 89 RNAi. See RNA interference (RNAi)
RNAse-free, 87 RNA-induced silencing complex (RISC), 71
specimen collection, 87 RNA-metabolizing enzymes, 36–37, 37t
total RNA, 87 RNase protection, 138
long noncoding, 71–72 RNases, 37t
messenger RNA, 27, 58 RNases H, 11b
methylation, 69 RNA-SSCP, 209b
microRNAs, 70 Robertsonian translocation, 187, 189f
molecular techniques for, 3 rRNA. See Ribosomal RNA (rRNA)
noncoding, 69–73 rSSCP, 209b
nucleolus, 28 RT-PCR, 378f
nucleosome, 65 RUO. See Research use only (RUO)
open reading frame, 31
operator, 61 S
operon, 61, 61f S1 nuclease, 18
polyA tail, 31 Safety Data Sheets, 467
polyadenylate polymerase, 59 Salting out, 83
polyadenylation signal, 29 Sample labeling, 137f
polyadenylic acid, 31 Sample preparation, 304–305
polycistronic, 29 Sanger sequencing. See Dideoxy chain termination (Sanger) sequencing
polymerases, 27, 28t, 35–36, 35b, 58, 58t Sarcomas, 370
polythymine, 31 Satellite plasmid DNA, 26
primers, 11b Scorpion primer/probes, 162, 163f
promoter, 28, 58 Screening, 428–429
556 Index
SDA. See Strand displacement amplification (SDA) sequencing ladder, 230

Secondary antibody, 123 software programs, sequence data, 235t
Secondary resistance mutations, 319 TIGR Assembler, 235t
Secondary structure, 42 Sequencing ladder, 230
Selenocysteine, 38b DNA synthesis, 228f
Selenoproteins, 38b reading of, 226f
Self-sustaining sequence replication (SSSR), 164–165 sequencing, RNA, 237–238
Self-transmissible plasmid, 26 Sequencing library, 239–240
Semiconservative, 9 Sequencing panels, 405
Semi-nested PCR, 157–158 Sequencing-based methods, 213–215
Sense strand, 59 allelic discrimination with fluorogenic probes, 214–215, 215f
Sensitivity control, 305, 457 sequence-specific PCR, 213–214
SeqScape, 235t Serological analysis, 427–430
Sequence complexity, 128 alloantibodies, 428
Sequence interpretation antihuman antibodies, 428
base calling, 232 complement-dependent cytotoxicity, 427
dye blobs, 232, 233f crossmatching, 429, 430f
mutations or polymorphisms, 234f cross-reactive epitope groups, 428
sequence quality, 233f HLA typing, 427–428
software programs, 232, 235t humoral sensitization, 428
Sequence of nucleotides, 4, 180 mixed leukocyte reaction, 429–430
Sequence targets, 307 panel reactive antibodies, 429
Sequence-based typing, 433–435, 433f, 434f private antibodies, 428
Sequence-specific oligonucleotide probe hybridization (SSOP), protein gel electrophoresis, 430
431–432, 431f public antigens, 428
Sequence-specific PCR (Sequence-specific primer PCR, SSP-PCR) screening, 428–429
factor V Leiden, 352f typing trays, 427
molecular analysis of the MHC, 432–433, 432f, 433f Serologically defined HLA specificities, 422t–424t
nucleic acid analyses, 213–214 SETS. See Swab Extraction Tube System (SETS)
target amplification, 156–157 Sex-linked disorders, 348
Sequence-specific primer amplification, 213f Sexually reproducing organisms, recombination in, 19–21
Sequencing DNA, 223–257 Shark tooth combs, 107
Assign, 235t Short interspersed nucleotide sequence (SINES), 261
bioinformatics, 248–250 Short tandem repeat TH01, 267f
bioinformatics terminology, 249t Short tandem repeats (STR), 261, 267, 268f. See also STR subjects
bisulfite DNA sequencing, 236–237 Sibling index, 277
BLAST, 235t Sibling tests, 277–278
consensus sequences, 248 Side chain, 38
cytosine residues, 238f Sigma factor, 35b
dideoxynucleotide, 226f Signal amplification, 172–174
direct sequencing, 224–235 branched DNA amplification, 172–173, 172f
electropherogram, 233f cleavage-based amplification, 173–174, 174f
Factura, 235t cycling probe, 174
FASTA, 235t hybrid capture assays, 173, 173f
fluorescent sequencing chemistries, 231f Signal-to-noise ratio, 132
GRAIL, 235t Signal transduction, 375b
Human Genome Project, 250–254, 250t, 251t, 253t Silencers, 63
IUB universal nomenclature, 248 Silent nucleotide substitution, 200
Basic Local Alignment Search Tool, 248 Silver-enhanced in situ hybridization (SISH), 373
hierarchal shotgun sequencing, 251, 252f Silver stain, 90, 110–111
for mixed bases, 250t Similarity, 249t
manual dideoxy sequencing, 225f Simultaneous replication, 10f
Matchmaker, 235t SINES. See Short interspersed nucleotide sequence (SINES)
Maxam-Gilbert sequencing, 224t, 225f Single nucleotide polymorphisms (SNPs), 289–291
next-generation sequencing, 238–248, 239b, 239f, 456 defined, 261
Phrap, 235t Human Haplotype Mapping project, 290–291, 290f, 291b
Phred, 235t types, 290t
Polyphred, 235t Single-gene disorders, 355–363
pyrosequencing, 235–236, 236f inheritance patterns, 346–349
SeqScape, 235t molecular basis, 349–355
Index 557
molecular methods, 350t SSSR. See Self-sustaining sequence replication (SSSR)

nonclassical patterns, 355–363 Staggered separation of the duplex, 16
Single-locus probe (SLP), 266, 266f Staining pattern, chromosomes, 185f
Single-strand break, 19 Standard curve, 457
Single-strand conformation polymorphism (SSCP), 207–209, 208f Standard precautions, 450
Single-stranded DNA, reannealing, 129f Standard tiling, 212, 212f
SISH. See Silver-enhanced in situ hybridization (SISH) Star activity, 114
Sizes of human chromosomes, 180t Sticky ends, 16
Slot blot, 133–134, 134f Stoffel fragment, 150
SLP. See Single-locus probe (SLP) Stop codon, 200
Sm proteins, 34b Storage diseases, 351t
Small interfering RNA, 70 Storage of reagents, 466
Small nuclear RNA, 33, 33t STR. See Short tandem repeat (STR)
Small RNAs, 70–71 STR analysis, 268–278
Snell, G., 418b STR locus information, 271t
SNPs. See Single nucleotide polymorphisms (SNPs) STR nomenclature, 270
Software programs, 235t STR TH01, 267f
Solid matrix RNA isolation, 89f STR typing by PCR, 266–278
Solid tumors, molecular abnormalities, 383t allelic ladders, 268
Solid-phase emulsion PCR, 166–167, 167f discrete allele systems, 268
Solid-phase isolation, 84–85, 84f gender identification, 270, 270f
Solution hybridization, 138–139, 138f internal size standards, 268
gel mobility shift assay, 139 microsatellites, 267
RNase protection, 138 microvariants, 267
solution hybridization, 138–139 mini-STR, 268
Somatic hypermutation, 388 short tandem repeats, 267
Southern blot STR analysis, 268–278
defined, 117 STR nomenclature, 270
example, 133f test results analysis, 270, 272, 272b, 272t, 273b
nitrocellulose, 117 Strand, 143
preparation of resolved DNA for blotting, 118–119 Strand displacement amplification (SDA), 169–170, 170f, 171f
probe, 117 Streptococcus pneumoniae, 311
restriction enzyme cutting and resolution, 117–118 Stringency, 128
single nucleotide polymorphisms, 262f Structural sequences, 43
Spa typing, 334–335 Stutter, 286–287
Specimen collection, 302–304, 448–450 Submarine gels, 106
Specimen handling, 447–451 Submetacentric chromosome, 184, 184f
collection tubes for molecular testing, 448–450 Substituted nucleotides, 7–8, 7f
contact precautions, 450 Substrate specificity, 14
criteria for accepting specimens, 447–448, 448f Subtelocentric chromosomes, 184f
cryotubes, 451, 453f Sugar-phosphate backbone, 8
holding and storage requirements, 451 Supercoiled DNA, 19
preanalytical error, 447 Supercoiled plasmids, 26, 26f
precautions in sample processing, 450 Suppressor tRNAs, 47b
standard precautions, 450 Surface amplification, 167, 168f
transmission-based precautions, 450 Surname test, 282
Specimen storage, 452t Susceptibility testing, 327
Specimen transport systems, 303t Swab Extraction Tube System (SETS), 303f
Spectral karyotyping, 193 SwissProt, 249t
Spectrophotometry, 92–93 SYBR green, 160
Spin columns, 155, 156f Syngeneic transplant, 284b
Splicesosome, 34b Synonymous changes, 425
Splicing, 31–33, 32f, 33b Synovial sarcoma translocation, 377–378
Split chimerism, 286b
Split specificities, 422 T
SSCP. See Single-strand conformation polymorphism (SSCP) T: IUB universal nomenclature, 248
SSOP. See Sequence-specific oligonucleotide probe hybridization Basic Local Alignment Search Tool, 248
(SSOP) hierarchal shotgun sequencing, 251, 252f
SSOP assay, 431f t(1;13), t(2;13), 378
SSP-PCR. See Sequence-specific PCR (SSP-PCR) t(8;14)(q24;q11), 400, 400f
558 Index
t(9;22)(q34;q11), 401–403, 401f, 402f, 403f analytic specificity, 454, 454t

t(11;14)(q13;q32), 398–399, 400f calibration curve, 457
t(14;18)(q32;q21), 397–398, 397f, 398f, 399f clinical sensitivity, 454t
t(15;17)(q22;q11.2-q12), 403–404, 404f clinical specificity, 454t
Tailed primers, 149, 149f controls, 457–458
Tandem repeat, 265f cut-off values, 458
Taq polymerase, 150 detection limit, 454t, 455
TaqMan, 160, 161f high-positive controls, 457
TaqMan probe, 162f internal controls, 457
TaqMan signal fluorescence, 162f linearity, 454t
Target, 143 low-positive controls, 457
Target amplification, 143–168 measurements, 454t
genomic amplification methods, 165–168 negative controls, 457
polymerase chain reaction, 143–164 positive controls, 457
transcription-based amplification systems, 164–165 precision, 454t
Target sequences, nucleic acid test, 307f quality assurance, 458–459
Targeted libraries, 241–242, 242f, 243f reference range, 454t
Tatum, Edward L., 23b reportable range, 454t
T-cell lymphoma, 406t reproducibility, 454t
T-cell receptor gene, 395f, 396f sensitivity control, 457
T-cell receptor gene rearrangements, 390–391, 390f, 390t, 391f, standard curve, 457
394–396, 395f, 396f Tetramethylethylenediamine (TEMED), 102
T-chronic lymphocytic leukemia, 406t Thermal cyclers, 151–152
TE buffer, 83b Thermometers, certified chamber, 460f
Telocentric chromosome, 184 Threshold cycle, 160
Telomere, 192 Thrombosis, 351t
Telomeric probe, 192, 193f, 194f Thymidine, 4
TEMED (tetramethylethylenediamine), 102 Thymine, 3, 14b
Template, 9, 143, 224 Thymine dimers, 14b
Teratocarcinomas, 370 TIGR Assembler, 235t
Terminal transferase, 14 Tiselius, Arne, 98b
Termination, transcription, 29 Tissue fixatives, 81t
Tertiary structure, 42–43 Tissue samples, 81
Test Tissue typing, 417–445, 418
allelic frequencies, paternity testing, 275–277 additional recognition factors, 437–438
analytical targets, molecular testing, 372 allografts, 418
avuncular, 277–278 bead arrays, serum antibody detection, 429f
bone marrow engraftment, 283–289 CDC assay example, 428t
calibrators, 456–457 CDC expression, 428t
controls, 457–458 crossmatching, antibodies, 427f
documentation of test results, 468–469 cytotoxicity, cells stained for, 428f
FDA approved, 455 DNA polymorphisms, 421f
FDA cleared, 455 graft-versus-host disease, 418
laboratory-developed, 451 HLA polymorphisms, 420–425
next-generation sequencing, 456 allele, 420, 421–422
nucleic acid amplification, 315t–317t haplotype, 420, 421f
parentage, 264–265, 265f HLA nomenclature, 420, 422–425
paternity, 276t HLA type, 420
performance. See Test performance KIR gene cluster, 438f
proficiency, 468 major histocompatibility complex, 418, 419t
quality assurance, 289f MHC disease association, 438–439
sibling, 277–278 MHC locus, 418–420
surname, 282 molecular analysis of MHC, 425–437
validation, 455 combining typing results, 436, 436t
in vitro analytical test, 465 coordination of HLA test methods, 437
Test performance, 451, 453–458 crossmatching, 427
amplification control, 457 DNA-based typing, 430–436
analyte measurement range, 454t HLA test discrepancies, 436–437
analytic accuracy, 454, 454t serological analysis, 425–437
analytic sensitivity, 454, 454t polypeptides, 420f
Index 559
reverse dot-blot SSOP, 431–432, 432f Types/structures of RNA, 29–35

SSOP assay, 431f small interfering RNA, 70
transplant evaluation, 439t small nuclear RNA, 33
Tissue-specific molecular testing, 372 small RNAs, 70–71
TMA. See Transcription-mediated amplification (TMA) transfer RNA, 33–34, 35b
Topoisomerase inhibitors, 19 Typing capacity, 336
Topoisomerases, 19 Typing method comparison, 336–337, 337t
Touchdown PCR, 155 discriminatory power, 337
tracrRNA, 115 reproducible, 337
Tracking dye, 108–109 typing capacity, 336–337
Traits, 3 Typing trays, 427
Trans factors, 60 Tyrosine-kinase activity, 372, 373f
Transcription, 27–29 Tyrosine-kinase inhibitors, 374
defined, 27, 58
elongation, 28–29, 28f, 58–59
factors, 64f
U
Unbalanced, 187
initiation, 28, 58
Ultraviolet light (uv), 462, 462f
post-transcriptional regulation, 64
Uniparental disomy, 362–363
regulation of epigenetics, 65–69
Uracil, 27, 27f
regulation of RNA synthesis at initiation, 60–64
Urogenital tract pathogens, 311–313
termination, 29, 59–60
Chlamydia trachomatis, 311
Transcription-based amplification systems, 164–165
Mycoplasma spp., 313
nucleic acid sequence-based amplification, 164–165
Neisseria gonorrhoeae, 311
self-sustaining sequence replication, 164–165
Treponema pallidum, 311–313
steps, 164f
transcription-mediated amplification, 164–165
Transcription-mediated amplification (TMA), 164–165 V
Transcriptome, 136b Vacuum transfer, 121, 121f
Transduction, 23 Validation, 306
Transfer RNA (tRNA), 33–34, 35b, 46 Vancomycin structure, 329f
Transformation, 23–25 Vancomycin-resistant S. aureus, 327f
Transforming factor, 24f, 25 Variable expressivity, 349
Translation, 46–51 Variable-number tandem repeat (VNTR), 261
amino acid charging, 46–47 Variant, 180
post-translational regulation, 64–65 Varicella zoster virus, 321
protein synthesis, 47–51, 50f V(D)J recombination, 387, 387f
Translocations, 186 Vertical gel apparatus, 108f
Transmembrane proteins, 40f Vertical gel electrophoresis, 98f
Transmission patterns, 346 VHL. See Von Hippel-Lindau syndrome (VHL)
Transmission-based precautions, 450 Viral load, 317
Transplant evaluation, 439t Viral load measurement, test performance, 318t
Transposon, 327 Viruses, 313–323
Treponema pallidum, 311–313 BK/JC viruses, 323
Trimming of nucleotides, 391 cytopathic effect, 313–314
Trinucleotide-repeat disorders, 362 DNA isolation, 80
Triploid, 346 DNA template, 149
Trisomy, 181 hepatitis C virus, 321–322
tRNA. See Transfer RNA (tRNA) herpes viruses, 320–321
tRNA charging, 46 human immunodeficiency virus, 314, 317–319
True negative, 305 human papillomavirus, 322
Tth polymerase, 150 mass spectrometry, 323
Tumor protein 53, TP53 (17p13), 378–379 mycology, 323–324
Tumor-specific molecular testing, 372 nucleic acid amplification tests, 315t–317t
Tumor-suppressor genes, 371 respiratory viruses, 322–323
t(X;18)(p11.2;q11.2), 377–378 Visualizing chromosomes, 184–186
Type I restriction enzymes, 15 4′,6-diamidino-2-phenylindole, 186
Type II restriction enzymes, 16, 16b, 16f C banding, 185b
Type III restriction enzymes, 15 G bands, 185, 185b, 186b
Types of gene mutations, 200–201 high-resolution banding, 186
Types of polymorphisms, 261 nucleolar organizing region staining, 186
560 Index
Visualizing chromosomes (cont’d) X-linked genes, 348

Q banding, 184–185, 185f X-linked recessive diseases, 348f
R banding, 185b xMHC. See Extended MHC locus (xMHC)
V-myc avian myelocytomatosis viral-related oncogene,
neuroblastoma-derived, 381 Y
VNTR. See Variable-number tandem repeat (VNTR) Y-STR, 278–282
Von Hippel–Lindau gene, VHL (3p26), 380–381 autosomal STRs, 278
V-Ros Avian UR2 Sarcoma Virus Oncogene Homolog 1 (ROS1) genotypes, 281t
Proto-Oncogene (6q22.1), 381 locus information, 280t–281t
VZV. See Herpes virus (VZV) matching with Y-STRs, 279, 281–282
discriminatory capacity, 279
W haplotype diversity, 279
Waddington, Conrad, 65 surname test, 282
Waldenström macroglobinemia, 406t paternal lineage test, 279
Warburg-Christian method, 93b purpose of, 268
Watson, James, 3, 44b
Wells, 106 Z
Western blot, 122–123, 123b, 127b, 127f, 133f Zamecnik Paul, 46–47
Whole chromosome paints, 193, 194f amino acid charging studies, 46
Whole-genome amplification, 165, 165f, 166, 166f ribosome studies, 47
Wollman, Elie, 23 Zimmerman, Steven B., 12b
Zinc finger motif, amino acid structure, 42b
X Zwitterions, 38
Xenogeneic transplant, 284b
X-linked, 346
QUICK VISUAL REFERENCE FOR COMMON
TECHNIQUES IN THE CLINICAL LABORATORY
120 140 160 180 200 220 240 260 280 300 320
LPLD5S818 D13S317 D7S820 D16S539
LPL D5S818 D13S317 D7S820 D16S539
vWA TH01 TP0X F13A01CSF1P0
■ Screening of 16 loci for informative short

tandem repeat alleles
■ Sequence-based typing result for HLA-A gene
■ Ethylene diamine tetra acetic acid (EDTA)

or acid citrate dextrose (ACD) tubes preferred ■ Electrophoresis capillary replacement and instrument
for molecular analysis of blood or bone marrow cleaning are examples of routine maintenance performed by
specimens laboratory personnel.
Continued from inside front cover

Molecular Diagnostics

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QUICK VISUAL REFERENCE FOR COMMON

TECHNIQUES IN MOLECULAR BIOLOGY

Threshold cycle (C)

■ Quantitative polymerase chain reaction (PCR)

Normal cell (diploid) Triploid Deletion

■ DNA methylation at cytosine residues detected by pyrosequencing

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Protein Probes 124

PYROSEQUENCING 235 QUALITY ASSURANCE 305

COLOR PLATE 4 Multicolor fluorescence in situ hybrid-

COLOR PLATE 2 Fluorescence in situ hybridization (FISH)

GCTGGTGGCGTA GCTTGTGGCGTAG CTACGCCACAAGC

HEALTH HAZARD FIRE HAZARD

COLOR PLATE 10 Biohazard stickers are required for cabi-

COLOR PLATE 12 National Fire Protection Association

Outline Transfer RNA

H2C CH CH2 H 2C CH CH2

is a left-handed helix with 12 bp per turn and Advanced Concepts

nucleosides, respectively. Azidothymidine (Retrovir, Growing strand

on their sugars. A nucleic acid chain grows by the attach- OH

growing chain (Fig. 1.6). Addition of nucleotides in this

phosphate end and a 3′ hydroxyl end. We refer to DNA O– P O P O P O–

HO In the process of replication, DNA is first unwound

Overall direction of replication

Primase is an RNA-synthesizing enzyme that catalyzes

A Pol I E. coli Recombination,

A T5 pol, T7 pol T5, T7 Replication

Polymerases Y Pol IV, Pol V E. coli Bypass

Leading strand DNA

a mismatch (e.g., A opposite C instead of T on the tem- 5′ 3′

(faithful copying of the template), and substrate speci-

After replication, distortions in the DNA duplex

TABLE 1.2 Types of Restriction Enzymes

Type Methylation Activity Binding Site Example Cut Site Example

I + AMeCN6G T Variable distance from binding EcoKI, CfrAI

II – GAATTC Within binding EcoR1, BglII

IIS – ACCTGC 1–20 bases from binding BfuAI

IIG + CTGAAG To one side or both sides of binding AcuI

III + CGAAT 25–27 bp 3′ to binding HinfIII

IV + GGGAC Asymetrical Bsplu11III

Eco RI SmaI Pst l

Type II restriction enzymes are those used most fre-

manipulate DNA in vitro,28 for instance, to make step-

What Mendel saw: What Mendel inferred:

RG RGrg RGrg RGrg RGrg

315 108 101 32

RG RRGG RRGg RrGG RrGg

Rg RRGg RRgg RrGg Rrgg

rG RrGG RrGg rrGG rrGg