MOLECULAR BIOLOGY – MIDTERM TOPIC 1

Mercado, Ada Audrey Nichole J. BMLS 10-2D

DNA AND RNA ISOLATION

Nucleic Acid Extraction: removal of nucleic acids (DNA and/or

RNA) from the cells in which they normally reside.

• Purpose: release nucleic acid from the cell for:

o Detecting a specific pathogen (bacteria and
viruses)
o Diagnosing disease and genetic disorders
o Other applications: forensics, paternity tests,
ancestry tracking, genetic engineering, vaccines,
hormones

Target Nucleic Acids: free from contamination with

macromolecules such as: proteins, carbohydrates, lipids, or
other nucleic acids
• Later Routine Laboratory Procedures of DNA Isolation
How Nucleic Acid is Removed from the Cell
o Solubility differences among chromosomal DNA,
plasmids, and proteins in alkaline buffers

1. Bust out the cell membrane I. Preparing the Sample:

2. Lysis of cell wall or cell membrane
Specimen Expected Yield
3. Lysis of nuclear membrane
Specimens Adequate for Analysis Without DNA
4. Nucleic acids come out from nucleus
Amplification
5. Purification
Blood¹ (1 mL, 3.5-10×10⁴ WBCs/mL) 50 – 200 µg
6. Determination of the concentration and purity of the sample
Buffy coat¹ (1 mL whole blood)
Isolation of DNA Bone marrow¹ (1 mL) 100 – 500 µg
Cultured cells (10² cells) 30 – 70 µg
• Friedrich Miescher (1869): isolated the nuclein (DNA)
Solid tissue¹ (1 mg) 1 – 10 µg
from the WBCs he obtained from the pus on collected
Lavage fluids (10 mL) 2 – 250 µg
surgical bandages in a nearby hospital. (Precipitation
Mitochondria (10 mg tissue, 10² cells) 1 – 10 µg
process)
Plasmid DNA, bacterial culture (100 mL 350 µg – 1 mg
o pus: composition of dead or alive bacteria,
overnight culture)
offending agent, or pathogen + WBC; product of
Bacterial culture (0.5 mL, 0.7 10 – 35 µg
immune response
absorbance units)
• Early Routine Laboratory Procedures of DNA Isolation
Feces⁵ (1 mg; bacteria, fungi) 2 – 228 µg
o Developed from density-gradient
Specimens Adequate for Analysis With DNA Amplification
centrifugation strategies
Serum, plasma, cerebrospinal fluid¹ 0.3 – 3 µg
▪ Meselson & Stahl (1958): demonstrate the
(0.5 mL)
semi-conservative replication of DNA
Dried blood (0.5 to 1 cm diameter spot) 0.04 – 0.7 µg
Saliva (1 mL) 5 – 15 µg
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

Buccal cells (500 mg) 3 – 50 µg presence of Tris

Bone, teeth (500 mg) 30 – 50 µg base, ethylene-
Fixed tissue** (5 – 10 × 10-micron 6 – 50 µg diaminetetraaceti
sections; 10 mm²) c acid (EDTA) &
Feces¹¹ (animal cells, 1 mg) 2-100 pg glucose.
* Yields are based on optimal conditions. Assays will vary in B. Viruses
yield and purity of sample DNA.
Viral DNA
* DNA yield will vary with WBC count. • Held within free viruses or integrated into the host
genome along with host DNA.
* DNA yield will depend on type and conditions of tissue.
• Cell-free specimens (plasma) will be used for viral
* DNA yield will depend on types and fungi will yield more or less detection
DNA. • May require concentration of viroids by centrifugation or
other methods
* DNA yield will depend on degree of cellularity.
• virions: basic structure of virus
* Dried blood yield from paper is less than from textiles.

* Mitochondrial DNA are attainable from hair shafts. C. Nucleated Cells in Suspension (Blood & Bone Marrow
Aspirates)
** Isolation of DNA from fixed tissue is affected by the type of
White Blood Cells (WBCs)
fixative used and the age of the preliminary handling of the
• Anticoagulants will be added to prevent clotting and will
original specimen.
also trap WBCs
¹* Cells in fecal specimens are subject to digestion and • Obtained from the blood plasma
degradation. • Purified of red blood cells (RBCs) & other components

A. Bacteria and Fungi: by either differential density-gradient centrifugation or

differential lysis
Gram- Some bacteria & Yeast, • Differential osmotic fragility of RBCs & WBCs
negative fungi with tough cell filamentous • Plasma & WBC: usually recovered for further analyses
bacteria walls fungi, & gram-
• Ficoll & RBC / Erythrocytes / Granulocytes: usually
positive
discarded but can be recovered as rich source of high-
bacteria
quality DNA
• Can be • Can be lysed by • Commercial
lysed by enzyme products reagents
high pH or mechanically designed for
(alkaline) grinding or by isolation of
and vigorously mixing DNA in
detergent with glass beads amplificatio
s (to crack them n
open and to break procedures
down the bonds (PCR)
present) D. Plasma

• Detergent (1% Exosomes

sodium dodecyl • Small vesicles (30 – 100 nm in diameter), which form by

sulfate) & strong imagination & budding from the inside of cellular

base (0.2 M endosome vesicles and are secreted by living cells

NaOH) in the • Contain nucleic acid and can be collected through

centrifugation
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

• Diagnostic & prognostic analyses purposes (liquid - can be accomplished using an organic mixture
biopsy) (phenol & chloroform)
• Solid-phase collection of nucleic acid - use a combination of high salt, low pH, and an
organic mixture of phenol and chloroform
o readily dissolves
E. Tissue Samples
o hydrophobic contaminants (lipids & lipoproteins)
Fresh Tissue Frozen Tissue Fixed, embedded
o collect cell debris & strips away most DNA-
tissue
associated proteins
• Dissociate • Dissociated • May be
d by by grinding deparaffinized
mincing the and by soaking in
tissue homogenizing xylene and
the tissue tissue is
rehydrated by
soaking it in
decreasing
concentrations
B. Inorganic Isolation Methods:
of ethanol
- does not include organic extraction step
• May be used
- sometimes called “salting out,” makes use of low-pH
directly
& high salt conditions to selectively precipitate
without
proteins, leaving the DNA in solution
dewaxing

Tissue Fixatives Influencing Nucleic Acid Quality

Fixative Relative Quality Average

of Nucleic Acid Fragment
C. Solid-Phase Isolation:
Fixative Size
- uses solid matrices (silica-based products) in the
Range (kB)
form of columns & beads to bind & hold DNA for
10% buffered Good 2.0 – 5.0
washing.
neutral formalin
Acetone Good 2.0 – 5.0
Zamboni’s Not as good 0.2 – 2.0
Clarke’s Not as good 0.8 – 1.0
Paraformaldehyde Not as good 0.2 – 5.0
Methacarn Not as good 0.7 – 1.5
Formalin-alcohol- Not as good 1.0 – 4.0
acetic acid
B-5 Less desirable <0.1
Carnoy’s Less desirable 0.7 – 1.5
Zenker’s Less desirable 0.7 – 1.5 III. Proteolytic Lysis of Fixed Material
Bouin’s Less desirable <0.1 • Paraffin-embedded specimens
o Dewaxed with xylene or other agents
o Rehydrated before nucleic acid isolation
II. DNA Isolation Chemistries
• Some tests (somatic mutation analyses)
A. Organic Isolation Methods:
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

o Microscopic examination of adjacent stained IV. Proteolytic Lysis of Fixed Material

serial section • Before lysis
Noninvasive Human DNA Isolation o Cells may be washed by suspension &
• Genomic DNA Purification from Hair centrifugation in saline / other isotonic buffers
o Simple alkaline lysis method: Hair with root is • Reagents for cell lysis
incubated (95ºC) for 10 minutes in NaOH buffer & the o Simple screens: detergents (SDS or Triton)
supernatant is subjected to DNA purification after o PCR amplification: mixture of Tris buffer &
centrifugation proteinase K
o Smooth chemical digestion method using
Dithiothreitol (DTT): Hair sample can be incubated
(56ºC for 2 hours with buffer containing Tris-HCl,
EDTA, NaCl, SDS, DTT, & proteinase K, followed by
gentle mixing and incubation (60ºC) for 2 hours or
until the hair has dissolved completely.
• Genomic DNA Purification from Saliva: cells found in
the saliva; exfoliated buccal epithelial cells & other cells
o Buccal swabs (cotton swabs / cytobrushes)
▪ Suspended in lysis buffer which includes Tris,
EDTA, SDS, & proteinase K
▪ Incubated (56ºC) for 1-3 hours until the tissue is
totally dissolved & DNA is extracted from the
solution
V. Rapid Extraction Methods
o Mouthwash Method
• PCR
▪ Samples from saline rinses need to be processed
/ frozen immediately after collection • Rapid lysis methods & DNA extraction / storage cards

▪ Alcohol-containing mouthwash must be used • Chelex

o Genomic DNA Purification from Urine Sample: o Cation-chelating resin that can be used for

▪ Urine Specimen simple extraction of DNA from minimal

• Inverted & swirled in a specimen cup to create samples.

a homogenous suspension of cells followed by o 10% Chelex resin beads is mixed with the
specimen & the cells are lysed by boiling.
the centrifugation
• Supernatant is removed & a dry pellet
containing cells is chilled (-20ºC) for 15
minutes followed by addition of lysis buffer
(Tris, EDTA, SDS, & proteinase K)
• Sample is incubated (56ºC) for 2h & then DNA
is extracted from the solution

VI. Isolation of Mitochondrial DNA

• Approaches
1. First, isolate the mitochondria by centrifugation
2. Isolate total DNA by hybridization or PCR.
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

• Tissue or cell pellets can be stored and/or

transported in preservative reagents

II. Preparation of Specimen Material

• Reticulocytes in blood & bone marrow samples:
Lysed by osmosis or separated from WBCs by
centrifugation
• Tissue: Kept frozen in liquid nitrogen / immersed in
buffer that will inactivate intracellular RNAses
• Bacterial & fungal RNA: Isolated by chemical lysis by
grinding in liquid nitrogen
Before isolation of RNA:
• Viral DNA: Isolated directly from the serum, plasma
• Strict precautions to avoid sample RNA degradation by culture medium, or other cell-free fluids by means of
ribonucleases (RNAses) specially formulated spin columns or beads
• RNAses must be eliminated or inactivated
o RNAses inhibitors: diethyl pyrocarbonate (DEPC) III. RNA Isolation Chemistries
added to water & buffers; vanadyl-ribonucleoside • Organic Isolation
complexes; and macaloid clays o Cell lysis
• Allocate a separate RNAse-free (RNF) are of the ▪ Detergent or phenol in the presence of high salt
laboratory (0.2 to 0.5 M NaCl) or RNAse inhibitors
▪ Guanidine isothicyanate (GITC) can also be used
Total RNA
▪ Strong reducing agents (2-mercaptoethanol) may
rRNA mRNA tRNA & snRNA also be added
(Ribosomal (Messenger (Transfer RNA & o Acid phenol: chloroform: isoamyl alcohol
RNAs) RNAs) Small Nuclear (25:24:1) solution efficiently extracts RNA
RNA
Most abundant Next most Also present
(80% - 90%) RNA abundant RNA
in all cells fraction (2.5% to
5%)

RNA Isolation

• Solid-phase Isolation
o Begins with similar steps as described for organic
extraction
o The strong denaturing buffer conditions must be
adjusted before application of the lysate to the
column
I. Specimen Collection
• RNA tests (array analysis or quantitative reverse
transcriptase PCR)
o RNA is stabilized from further metabolism or
degradation after collection
• Specialized collection tubes
o To stabilize RNA immediately upon draw
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

• The quality of RNA from fixed, paraffin-embedded tissue

will depend on the fixation process and handling of the
specimen before fixation
• Commercial reagent sets are available for extraction of
RNA from fixed-tissue specimens
• Specialized reagents or spin columns for removal of
genomic DNA contamination are included in some
systems
• Automated isolation systems have reagent kits and
cartridges

Fixative Relative Quality of Average Fragment

Nucleic Acid Fixative Size Range
IV. Yield of RNA from various specimen sources
(kB)
• 1 million eukaryotic cells or 10-50 mg of tissue = 10 µg of
10% buffered Good 2.0 – 5.0
RNA
neutral formalin
• Yield of RNA from biological fluids will depend on the
Acetone Good 2.0 – 5.0
concentration of microorganisms or other target
Zamboni’s Not as good 0.2 – 2.0
molecules present in the specimen
Clarke’s Not as good 0.8 – 1.0
Specimen Expected
Paraformaldehyde Not as good 0.2 – 5.0
Yield
Methacarn Not as good 0.7 – 1.5
Blood¹ (1 mL, 3.5-10×10⁴ WBCs/mL) 1 – 10 µg
Formalin-alcohol- Not as good 1.0 – 4.0
Buffy coat¹ (1 mL whole blood) 5 – 10 µg
acetic acid
Bone marrow¹ (1 mL) 50 – 200 µg
B-5 Less desirable <0.1
Cultured cells (10² cells) 50 – 150 µg
Carnoy’s Less desirable 0.7 – 1.5
Buccal cells (1 mg) 1 – 10 µg
Zenker’s Less desirable 0.7 – 1.5
Solid tissue¹ (1 mg) 0.5 – 4 µg
Bouin’s Less desirable <0.1
Fixed tissue¹ (1 mm³) 0.2 – 3 µg
Bacterial culture¹ (0.5 mL, 0.7 absorbance 10 – 100 µg
units) VI. Isolation of polyA (Messenger) RNA
*Specimen handling especially affects RNA yield isolation of • RNA often required for analysis: mRNA (2.5% - 5% of
polyA RNA will result in much lower yields. the total RNA yield)
• Single-stranded oligomers of thymine or uracil
*RNA yield will depend on WBC count.
immobilized on a matrix resin are used
*RNA yield will depend on type of cells and the conditions of cell o Approximately 30-40 ng of mRNA can be obtained
culture. from 1 µg of total RNA

*Liver, spleen, and heart tissues yield more RNA than brain,
lung, ovary, kidney, or thymus tissues.

*Isolation of RNA from fixed tissue is especially affected by the

type of fixative used and the age and the preliminary handling of
the original specimen.

*Different bacterial types and fungi will yield more or less RNA.

V. Proteolytic Lysis of Fixed Material

MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

Measurement of Nucleic Acid Quality & Quantity A = Absorbance

∈ = molar absorptivity (L/mol-cm), 50 for dsDNA, 40 for
RNA
b = path length (cm)
c = concentration (mg/L)
• Absorbance is directly proportional to the
concentration of the nucleic acid in the sample
• Beer-Lambert Law: A= ∈bc
o ∈ = molar absorptivity (L/mol-cm), 50 for dsDNA,
40 for RNA
Electrophoresis Spectrophotometry
Separation of particles Technique that uses light Using absorptivity, as a conversion factor from
through a solution or matrix absorption to measure the optical density to concentration:
under the force of an concentration of an analyte in
At 260 nm,
electric current a solution
Fluorometry Microfluidics 1 optical density unit (or absorbance unit)

Measurement of emitted Science & technology of = 50 mg/L (or 50 µg/mL) of dsDNA & 40 µg/mL of RNA
fluorescent light systems that process or To determine concentration:
manipulate small amounts of
Spectrophotometer reading in absorbance units ×
fluid (10-9 to 10-18L), using appropriate conversion factor
channels measuring from tens
If the DNA / RNA preparation require dilution before
to hundreds of micrometers
spectrophotometry, to determine concentration:

Absorbance reading x conversion factor x dilution

factor

Example 1:

A DNA preparation diluted 1/100 yields an absorbance

reading of 0.200 at 260 nm. What is the concentration
of DNA in µg/mL?

To obtain the concentration in µg/mL

I. Electrophoresis
0.200 absorbance units x 100 = 1,000 µg/mL
• DNA and RNA can be analyzed for quality (detection &
size analysis by resolving an aliquot of the isolated The yield of the sample is calculated using the volume
of the preparation. If the DNA was eluted /
sample on an agarose gel
resuspended in a volume of 0.5 mL in the case
• Uses an electric current to propel charged illustrated previously, the yield would be:
biomolecules through a porous gel matrix at a rate that
1,000 µg/mL × 0.5 mL = 500 µg
is the function of the charge, size, and shape of the
Example 2:
molecules
• Fluorescent dyes used: ethidium bromide, SybrGreen An RNA preparation diluted 1/10 yields an absorbance
reading of 0.500 at 260 nm. What is the concentration
I, silver stain
of DNA in µg/mL?
• Appearance of DNA on agarose gels depends on the
To obtain the concentration in µg/mL:
type of DNA isolated
0.500 absorbance units × 40 µg/mL
II. Spectrophotometry Per absorbance units × 20 = 200 µg/mL
• Nucleic acids absorb light at 260 nm through adenine
The yield of the sample is calculated using the volume
residues of the preparation. If the DNA was eluted /
• Beer-Lambert Law: A= ∈bc
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

resuspended in a volume of 0.2 mL in the case (iii) 3 0.398 0.174

illustrated previously, the yield would be:
For each sample based on the A260 / A280 ratio, is each
200 µg/mL × 0.2 mL = 40 µg
preparation suitable for further use? If not, what is the
• May also be used to estimate the purity of nucleic contaminating DNA?
acid
Solution: Get the A260 / A280 ratio for each sample
• Detection of contaminants: reading the
concentration over a range of wavelength i. 0.419 / 0.230 = 1.82 – suitable
• Indication of contamination: absorbance over ii. 0.258 / 0.225 = 1.15 – not suitable ; protein
background at any wavelength other than the iii. 0.398 / 0.174 = 2.29 – may be suitable if RNA does
A260 maxima of the nucleic acid. not interfere with the subsequent assay
o Absorbance of DNA at 260 nm = 1.6 to 2.00
times more than the absorbance at 280 nm • UV Spectrophotometry
o RNA = 2.0 to 2.3 o Standard nucleic acid quantitation
o If the 260 nm / 280 nm ratio is <1.6, the o Nucleic acid sample is placed into quartz cuvette,
nucleic acid preparation may be which is then placed inside the UV
contaminated with unacceptable amounts of spectrophotometer
protein & not of sufficient purity for use o UV light passed through the sample at a specified
• Most likely contaminant: protein (absorbs light at path length, & the absorbance of the sample at
260 nm through the aromatic tryptophan & specific wavelengths is measured
tyrosine residues) o Does not require additional reagents / incubation
time
Common Contaminants and Their Wavelengths of Peak
• NanoDrop Spectrophotometry
Absorbance
o Similar in principle with the previous, but has many
Wavelength (nm) Contaminant
additional capabilities:
230 Organic compounds
▪ Functions by combining filter optic technology
270 Phenol
& natural surface tension properties
280 Protein
▪ Accompanied by special software to enable
>330 Particulate matter
analysis of signal from small quantities of
sample

• Absorbance of DNA at 260 nm = 1.6 to 2.00 times ▪ Displays the entire absorbance spectrum of the

more than the absorbance at 280 nm. sample in graphical form – allows detection of

• RNA = 2.0 to 2.3 contaminants

• If the 260 nm / 280 nm ratio is <1.6, the the nucleic ▪ Capable of determining a wide range of sample

acid preparation may be contaminated with concentrations w/o requiring serial dilutions

unacceptable amounts of protein & not of sufficient III. Fluorometry (Fluorescent Spectroscopy)

purity for use • Measures fluorescence related to DNA

concentration in association with DNA-specific
Example: fluorescent dyes

Three DNA preparations have the following A260 and A280 • Early methods: 3,5-diaminobenzoic acid 2HCl

readings: (DABA), combined with alpha-methylene aldehydes

(deoxyribose) to yield a fluorescent product
Sample OD260 OD280
• Modern methods: DNA-specific dye Hoechst 33258

(i) 1 0.419 0.230 combined with adenine-thymine base pairs in the

minor grove of the DNA double helix
(ii) 2 0.258 0.225
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

o Fluorometric determination of DNA

concentration down to 200 ng DNA/mL
• Other DNA-specific dyes
o PicoGreen: detection down to 25 pg/mL
concentrations
o OilGreen: detection down to 100 pg/mL of
ssDNA
• RNA: SybrGreen II RNA gel stain is used

Absorption and fluorometry readings may not always agree

• These methods recognize different targets

o Single nucleotides do not bind to fluorescent dyes,
but they can absorb ultraviolet light
o Absorption measurements do not distinguish
between DNA & RNA
o Deciding which instrument to use is at the discretion
of the laboratory:
▪ Most use spectrophotometry because the
samples can be read directly without staining
or mixing with dye
▪ Methods requiring accurate measurements of
low amounts of DNA/RNA (in the range of 10
to 100 ng/mL), fluorometry may be preferred
IV. Microfluidics
• Sample is applied to a multiwell chip & then moves
through microchannels across a detector
• Instrument software generates image in
electropherogram (peak) or gel (band) configurations
• RNA integrity number: quantification estimate for RNA,
determined as a standard measure of RNA integrity
• Uses a minimal volume of sample (as low as 1 µL) &
can test multiple samples simultaneously
• Useful for analysis of studies on small TNAs
(microRNAs) in eukaryotes & gene expression in
bacteria.
Molecular Biology and Diagnostics
ANALYSIS AND
CHARACTERIZATION OF
NUCLEIC ACIDS AND
PROTEINS

Nucleic Acid/ Protein Analysis

➔ Involves isolation and
characterization of DNA or RNA or
protein sample of interest for use in
applications.

ANALYSIS AND CHARACTERIZATION OF

NUCLEIC ACIDS AND PROTEIN

1. Restriction enzyme mapping of

DNA.
2. CRISPR enzyme systems Uses and Applications
3. Hybridization technology
● Gives an idea of genome
sequence.
● Used to map genomes when
1. Restriction Enzyme Mapping of sequence information is unkown.
DNA ● Can be used to introduce primers
for sequencing.
➔ Involves restriction enzyme
● Used for ID and characterization of
◆ Restriction enzymes such as
naturally-occuring plasmids and to
endonucleases.
engineer the construction of
◆ type II is commonly used, with
recombinant plasmids.
4-6 bp recognition or
restriction sites.
● Binding or cutting
➔ Restriction enzyme digestion of DNA
sites on DNA.
fragment/region = pattern of
◆ Used to identify the position
fragments
of restriction sites in DNA
◆ Used to identify that DNA
fragments in relation to each
other.
◆ Developed using small Restriction Fragment Length
circular bacterial plasmids. Polymorphisms RFLPs

➔ Resulting differences in the size or

number of restriction fragments.

MARTINEZ, ABIGAIL B. BMLS 2G

 ➔ Useful in edpidemiologic studies.
➔ The basis of the first
molecular-based human ID and
mapping methods.
➔ Clinical analysis of structural
changes in chromosomes
associated with disease.

2. CRISPR Enzyme Systems

➔ LOCATION: archea and bacteria. PROBE
➔ FUNCTION: guides a common
enzyme to specific sites determined ➔ ss fragment of nucleic acid/protein
by RNA components. with a detectable signal that
➔ USE: manipulation of both DNA specifically binds to complementary
sequence and RNA expression. sequences/target protein.
➔ CRISPR stands for clustered regularly ➔ PURPOSE: identify one or more
interspaced repeats. sequences of interest with a large
◆ Protective system using the amount of nucleic acids.
invading DNA to target itself. ➔ OTHER USED: modified nucleic acid,
◆ Encodes an endonuclease: such as peptide nucleic acids and
● CRISPR-associated locked nucleic acids.
protein (Cas)
➔ TYPES:
A. DNA Probes
◆ Type I & II - target dsDNA ➔ Sources:
◆ Types III - targets ssDNA & ◆ EARLY METHODS: fragment
RNA of gene was cloned on
bacterial plasmid — isolated
by restriction enzyme
3. Hybridization Technologies digestion and gel purification
— labeling and denaturation
➔ A reaction that is used to analyze the
of fragment — applied to
nucleic acid content of an unknown
southern/northern blot.
sample.
◆ Isolation of sequence of
➔ Formation of H bonds between 2
interest from viral genomes.
complementary strands of nucleic
◆ In vitro organic synthesis of
acid.
predetermined sequence —
➔ IN HYBRIDIZATION TECHNIQUE:
only for short, oligometric
◆ First strand is unlabeled.
probes.
◆ First strand is labeled (with
◆ Synthesized using PCR
isotopes/fluorescence) =
probe
➔ The probe is constructed so that it
has a complementary sequence to
the targeted gene.

MARTINEZ, ABIGAIL B. BMLS 2G

 ◆ IN ORDER THE PROBE TO ◆ MONOCLONAL
BIND: target nucleic acid has ANTIBODIES: products of a
to contain the sequence of single clone of plasma B
interest. cells.
◆ DENATURATION OF dsDNA
PROBED BEFORE USE: PROBE LABELING
● Heating the probe (95
degree celsius, 10-15 ➔ Must generate a detectable signal for
minutes) in visualization of the probe binding to
hybridization solution. the target fragments on a membrane.
● Treating with 50% ➔ EARLIER LABELING: radioactive
formalin/2x SSC at a 32^P
lower temperature for ➔ PRESENT LABELING:
a shorter time (75 nonradioactive labels/tags (biotin &
degree celsius, 5-6 digoxigenin).
minutes). ➔ THREE BASIC METHODS FOR DNA
PROBE LABELING:
B. RNA Probes ◆ End labeling
◆ Nick translation
➔ SOURCE: ◆ Random priming
◆ Transcription from a synthetic
DNA temperature in vitro. DESIGN & OPTIMAL HYBRIDIZATION
◆ Predesigned systems OF PROBE
commercially available.
➔ SOUTHERN BLOT: coding strand ➔ Determine the specificity of the
transcript. results
➔ NORTHERN BLOT: ➔ LONGER PROBES: 500-5000bp
◆ Antisense transcript ◆ Greater specificity — less
◆ Labeled with affected by points.
radioactive/modified ◆ mutations/polymorphisms
nucleotides for production of ◆ Difficult and expensive to
signal. synthesize.
➔ SHORTER PROBES: less than 500bp
C. Other Nucleic Acid Probe Types ◆ Less specific
◆ Higher chance of being
➔ Synthetic and resistant to nucleases repeated randomly in
◆ Peptide nucleic acid probes unrelated religions of the
◆ Locked nucleic acid probes genome.
◆ Unlocked nucleic acid probes ◆ Ideal for mutational analysis.

D. Protein Probes SOUTHERN BLOTS

➔ WESTERN BLOT ➔ The procedure was first reported by
◆ POLYCLONAL ANTIBODIES: Edwin Southern.
products of a generalized ➔ Detects specific DNA sequences.
response to a specific ➔ PROCEDURE:
antigen. ◆ Genomic DNA is isolated and
cut with restriction enzymes.

MARTINEZ, ABIGAIL B. BMLS 2G

 ◆ Fragments are separated by ● Denhardt solution
gel electrophoresis and then (ficoll, polyvinyl
transferred to a solid support pyrrolidone, bovine
(nitrocellulose). serum albumin)
◆ DNA fragments are exposed ● Salmon sperm DNA
to a labeled probe ● SDS, 0.01% may also
(complementary to DNA or be included with
RNA) that is complementary formamide.
to the region of interest. ◆ Membrane is exposed to the
buffer at the optimal
TYPES OF MEMBRANES hybridization temperature for
30 minutes several hours.
1. Nitrocellulose - bind 70-150 ◆ Sample is ready for
microgram of nucleic acids per cm hybridization with the probe.
squared ● Allow visualization of
2. Nylon the specific gene or
3. Cellulose modified with a dimethyl region of interest.
aminoethyl
4. Carboxymethyl (CM) chemical group SOUTHERN BLOT APPLICATIONS:
5. Polyvinyl difluoride (PVDF) - proteins
1. ID of the single gene in a pool of
TRANSFER METHODS DNA fragments.
2. Gene mapping
1. Capillary transfer 3. Analysis of genetic patterns of DNA.
2. Electrophoretic transfer 4. Detection of specific DNA sequences
3. Vacuum transfer in a genome.
5. Study of gene deletions,
duplications, and mutations that
cause various diseases.
6. Detection of genetic disease and
PREHYBRIDIZATION cancer.
7. Detects the presence of a gene
➔ Required the following DNA family in a genome.
immobilization to prevent the probe 8. DNA fingerprinting such as forensic
from binding to the nonspecific tests, paternity testing, and sex
sites on the membrane surface: determination.
◆ Incubation of membrane in
the same buffer in which the NORTHERN BLOT APPLICATIONS:
probe will subsequently be
introduced/ in a specially ● Designed to investigate RNA
formulated prehybridization structure and quantity:
buffer. 1. Levels of gene expression
◆ PREHYBRIDIZATION (transcription from DNA) and
BUFFER COMPOSITION: stability.

MARTINEZ, ABIGAIL B. BMLS 2G

 2. RNA structural abnormalities ➔ FACTORS AFFECTING
resulting from aberrations in STRINGENCY:
synthesis/processing ◆ Temperature of hybridization.
(alternative splicing). ◆ Salt concentration of the
● Sample is cut out from the gel and hybridization buffer.
soaked in ammonium acetate to ◆ Concentration of the
remove denaturant and will be denaturant (formamide) in
stained buffer.
● STAINED: acridine orange or ◆ Length and nature of the
ethidium bromide probe sequence.
➔ Long probes with the higher
WESTERN BLOT percent of G and C bases will bind
under more stringent conditions
➔ IMMOBILIZED TARGET: protein than shorter probes with higher
➔ SDS-polyacrylamide gel percent of A and G bases.
electrophoresis (SDS-PAGE):
resolves proteins according to ESTIMATING THE IDEAL
molecular weight at 5%-20%
HYBRIDIZATION CONDITIONS
concentrations.
➔ Isoelectric focusing gels (IEF)/tube ➔ Calculation of the melting
electrophoresis: resolves proteins temperature of the probe sequence.
according to charge. ◆ Express the amount of the
➔ OTHER MEMBRANES USED: PVDF energy required to separate
and anion (DEAE) or cation (CM) the hybridized strands of
exchange cellulose. given sequence
➔ SIGNALS: chemiluminescent or color ● Half of the sequence
signals is ids and half is ss
➔ FORMULA FOR dsDNA:
HYBRIDIZATION CONDITIONS,
STRINGENCY
➔ Combination of conditions under
which the target is exposed to the ➔ FORMULA FOR SHORT PROBES
probe (14-20 BASES):
➔ HIGH STRINGENCY CONDITIONS:
◆ High hybridization temp + low
concentration of salt in the ➔ Hybridizations are generally
buffers performed in hybridization
◆ Probe will not bind to its bags/glass cylinders.
target. ➔ RECOMMENDED VOLUME FOR
➔ LOW STRINGENCY CONDITIONS: HYBRIDIZATION BUFFER: approx.
◆ Low hybridization temp + a 10 mL/100 cm^2 of membrane
high concentration of salt in surface area.
the buffer ➔ Shorts probes (less than 20 bases)
◆ Probe will not bind to can hybridize in 1-2 hrs
unrelated targets.

MARTINEZ, ABIGAIL B. BMLS 2G

 ➔ Long probes (greater than 1000
bases) can hybridize in 16 hrs or 4. Array-based Hybridization
more.
➔ Inert polymers (dextran, sulfate, A. DOT/SLOT BLOTS
polyethylene glycol, or polyacrylic
acid) accelerate for longer probes ➔ Applied to expression, mutation and
(greater than 250 probes). amplification/deletion analyses.
➔ Target DNA/RNA is deposited
DETECTION SYSTEM directly on the membrane by means
of various devices (vacuum systems
➔ Whether the probe has bound to the and pipette for few samples).
immobilized target. ➔ Performed on cloned plasmids, PCR
➔ RNA/DNA Probe Labeled with products, selected mRNA selections
Radioactive Phosphorus Atoms:
◆ Unbound probe is washed off DOT BLOT SLOT BLOT
and the blot is exposed to
light-sensitive film to detect ● Target is ● Target is
the fragments that are deposited deposited in
hybridized to the radioactive in a circle or an oblong
probe. dot. bar.
➔ Indirect Nonradioactive Detection ● Useful for ● Useful for
System: multiple accurate
◆ Unbound probe is washed qualitative quantificatio
away and anti-digoxigenin analyses n by
antibody/streptavidin is where densitometr
added to bind labeled probe many y scanning.
target complex targets are
◆ Membrane is bathed in a being
solution of a substrate compared
(dioxane/tetrazolium dye (mutational
preservatives) producing screening)
chemiluminescent
➔ BASELINE FOR INTERPRETATION:
signal/color signals.
negative control —DNA of equal
INTERPRETATION OF RESULTS complexity but without the target
sequence.
➔ Visualization of a “band” on the ➔ Amplification/normalization control is
membrane/film. also included.
➔ ANALYSIS OF BANDS:
presence/absence/location in the B. GENOMIC ARRAY TECHNOLOGY
lane
➔ Type of hybridization analysis allows
➔ SOUTHERN BLOT: comparing
simultaneous study of a large
restriction fragment lengths of
number of targets.
selected regions in different samples.
➔ APPROACHES:
➔ NORTHERN BLOT: amount of gene
◆ Macroassays
expression is determined relative to
◆ Microassays
the internal control

MARTINEZ, ABIGAIL B. BMLS 2G

 ◆ High-density oligonucleotide ➔ Target probes immobilized on chips
arrays are hybridized with labeled mRNA
◆ Microelectronic arrays from treated cells/different cell types.
➔ Measure transcript/protein
MACROASSAYS production relative to a reference
control for each target gene isolated
➔ REVERSE DOT BLOT: immobilized
from unrelated/normal specimens.
probe is now effectively the target
and the labeled specimen
COMPARATIVE GENOME
◆ DNA, RNA, proteins are
actually the probes
HYBRIDIZATION
➔ DETECTION OF HYBRIDIZATION: ➔ Designed to test DNA.
radioactive/chemiluminescent signals ➔ Used to screen the genome/specific
➔ READING OF HYBRIDIZATION: by genomic loci for deletions and
eye or phosphorimager amplifications.
➔ ANALYSIS: signal intensity from test ➔ Genomic DNA is isolated, fragments,
“spots” was compared to control and labels for hybridization on the
samples spotted on duplicate chip.
membranes. ➔ Can be performed on fixed tissue
and limiting samples.
MiCROASSAYS ➔ SAMPLE PREPARATION FOR
➔ Glass substrates are used for the ARRAY ANALYSES: requires
production of arrays. fluorescent labeling of the test
➔ Improved spotting and has the ability samples.
to deposit very small target spots. ➔ READING MICROARRAYS: requires
➔ Automated deposing systems a fluorescent reader and analysis
(arrayers). software.
◆ Can place less than 80,000 ➔ REPORTING OF RESULTS: relative
spots on the glass substrate. amount of the reference and test
◆ Pen-type and ink-jet signals.

PHOTOLITHOGRAPHY TECHNIQUE BEAD ARRAY TECHNOLOGY

➔ High-density oligonucleotide array ➔ Probes are immobilized on beads,

➔ Deposit target by DNA synthesis allowing hybridization of the targets
directly on the glass/silicon support. in the bead suspension.
➔ Uses sequence information to design ➔ Multiple suspensions can be tested
oligonucleotides at designated simultaneously.
positions on the chip. ➔ Beads are color-coded with a
➔ Used for mutation and polymorphism particular shade of red fluorescent
analysis, DNAmethylation analysis, dye.
and sequencing. ➔ Sample is labeled with a green dye.
➔ Used for protein and nucleic acid
EXPRESSION ARRAYS targets.
➔ Available for infectious diseases and
➔ For gene expression analyses. tissue typing.

MARTINEZ, ABIGAIL B. BMLS 2G

5. Solution Hybridization
➔ Neither the probe nor the target is
immobilized.
➔ Probes and targets bind in solution,
followed by resolution of the bound
products.
➔ To measure mRNA expression
➔ VERSIONS:
◆ RNAseprotection (S1
mapping): labeled probe
probe is hybridized to the
target sample in solution.
◆ Capture of DNA probe: RNA
target hybrids on a solid
support/beads: uses 2
probes (capture probe and
detection probe.

● HYBRIDIZATION METHOD
ADVANTAGE: direct analysis of
nucleic acids at the sequence level
without cloning of target sequences.
● SIGNIFICANCE TO CLINICAL
APPLICATIONS: direct recovery of
molecular genetic information from
routine specimen types.
● Widely varying modifications of the
basic methods have been and will be
developed for clinical and research
applications.

MARTINEZ, ABIGAIL B. BMLS 2G

MOLECULAR BIOLOGY AND DIAGNOSTICS: LARGE NA FRAGMENTS
LABORATORY NOTES
- May move rapidly in large pore size
RESOLUTION AND DETECTION OF (Low gel concentration)
ELECTROPHORESIS - May move slowly in small pore size
(High gel concentration)
GEL SYSTEMS AND BUFFER SYSTEMS
SMALL NA FRAGMENTS
- May move rapidly in large pore size
ELECTROPHORESIS
and small pore size.
- Movement of molecules in electric
To separate them, it should be in high gel
current.
concentrations.
Purpose: Separate molecules by size and
• PAPER
charge.
• TUBES (ISOELECTRIC FOCUSING)
• PROTEIN • SLAB GELS – Horizontal, Vertical
• NUCLEIC ACID • CAPPILLARIES
Remember: anions travel to the anode and GELS SYSTEMS
cations travel to the cathode.
- Provide resistance to the movement
of molecules under the force of
electric current forming bands.
TERMS:
BANDS
• CATHODE – The negative pole.
• ANODE – The positive pole. • The movement is impeded in the gel
• ANION – Negatively charged and will form it.
molecule. • Dependent to the speed of
• CATION – Positively charged migration.
molecule.
The concentration of the gel/buffers will
Nucleic Acids (NA) moves from negative to affect the resolution of fragments of
positive pole. different size ranges.

- NA sugar phosphate backbones are CRITERIA FOR A GOOD GEL:

negatively charges.
1. Unaffected by electrophoresis
When DNA is applied to a macromolecular 2. Simple to prepare
case or gel, its migration under the 3. Amenable to modification
pull of current is impeded.
Types of gel:
• AGAROSE
• POLYACRYLAMINDE
• COMPOSITE AGAROSE-
POLYACRYLAMIDE
AGAROSE GEL BUFFERS – Carry the current and protect
samples during electrophoresis.
• Polysaccharide polymer of
agarobiose • Composed of weak acid+ its
• Extracted from seaweeds conjugate base
• Component in agar • Uncontrolled Ph may damage the
• Usage: Powder is suspended in sample
buffer, heated and poured into • High concentration means higher
mold. conductivity, thus we need to lower
• Formulations: the voltage

- small DNA (50-500BP)-2- FOR DNA:

3% 1. TRIS BORATED EDTA (TBE)
- large DNA (2000-50000BP) 2. TRIS ACTETATE EDTA (TAE)
– 0.5-1% 3. TRIS PHOSPHATE EDTA (TPE)
• Easier casting
• Gel is negatively charged – thus FOR RNA:
surrounded by buffers 1. 10nM SODIUM PHOSPHATE
• Cooled 55-65C 2. MOPS BUFFER
• Usually on horizontal setups
BUFFER ADDITIVES
POLYACRYLAMIDE GEL • FORMAMIDE + HEAT + BLOCK
• Synthetic HYDROGEN BONDING SITES
• Higher resolution than agarose • UREA + HEAT = PREVENT
• Mobility can depend on DNA FOLDING
sequence (agarose do not, solely on Denaturing agents prevents
size only. hydrogen bonds and formation of
• For very small DNA, SSDNA, RNA, HETERODUPLEXES – FOLDED
proteins SSDNA/RNA forming HAIRPIN-LIKE
• Acrylamide + methylene structures that will impair
bisacrylamide MIGRATION.
• Forms: Powdered form (neurotoxic),
ELECTRECTROPHORESIS SETUP
commercial solutions, and
- Sample introduced into wells (holes)
performed gels.
at the top of the gel, which is made
• Usually in vertical setups
possible by combs.
• Requires a catalyst
-polymerization catalysts:
*AMMONIUM PERSULFATE
(APS) + N, N, N’, N’-
TETRAMETHYLENEDIAMINE (TEMED)
* LIGHT ACTIVATION
 CONTOUR- Alternating
CLAMPLED polarity in an
ELECTROPHORESIS COMBS:
HOMOGENOUS electrode array;
TYPE DESCRIPTION ELECTRICAL FIELD DNA molecules
Regular - WELLS SEPARATED (CHEF) as large as 2Mb
combs BY AN “EAR” OF THE can be well
GEL. separated.

- placed on TOP of ROTATING GEL Rotating gel with

the gel. ELECTROPHORESIS fixed poles;50kb
Houndstooth/ -WELLS IMMEDIATELY (RGE) to 6000 KB
sharkstooth ADJACENT
PFGE APPLICATIONS
-Placed upside-down,
- Molecular typing and identification
after polymerization
of pathogens
is placed side-down
- Gold standard method of some
at top.
bacterial ids
-Genotyping for antibiotics
resistance of:
-Staphylococcus aureus
PULSE FIELD GEL ELECTROPHORESIS -Pseudomonas aeruginosa
-Acinetobacter baumannii
- For very large DNA fragments (50k to -Mycobacterium tuberculosis
250 + kBP) - Epidemiological studies
- Pulses of current applied to the gel - Relationship of strains of same
in alternating dimensions enhance species
migration. - DNA fingerprinting of viruses

PULSE-FIELD GEL ELECTROPHORESIS TYPES: CAPILLARY ELECTROPHORESIS

TYPE DESCRIPTION - Separates solutes by charge/mass
FIELD INVERSION Alternating ratio
GEL positive and - Separated nucleic acids, metals,
ELECTROPHORESIS negative poles, anions, carbohydrates,
(FIGE) provides good pharmaceuticals
resolution - Thin glass (fused silica) capillary 30-
(>800kb) 100cm X 25-100um internal
diameter
TRANSVERSE Transverse angle - Linear or cross-linked
ALTERNATIVE GEL reorientation of polyacrylamide or other linear
ELECTROPHORESIS poles in a polymers used for sieving
(TAFE) vertical gel. - Separation based on size
More rapid, sensitive and automated than
slab gels
• Run at higher charge per unit area
• Electrokinetic injection for DNA
samples

RUNNING A GEL
➢ Use the proper gel concentration for
sample size range.
• 0.5-5% AGAROSE
• 3.5-20% POLYACRYLAMIDE

➢ Use the proper comb (well) and size

➢ Load sample mixed with tracking dye
(dye + density agent)

FLUORESCENT DYES
➢ Detect bands by staining
during or after
electrophoresis
• ETHIDIUM BROMIDE
– DSDNA
• SyBr green/gold –
SSDNA/RNA
• SILVER STAIN –
SSDNA/RNA/PROTEINS
MOLECULAR BIOLOGY AND *WHITE TOP – BROMOPHENOL BLUE
DIAGNOSTIC LABORATORY NOTES
*RED TOP – XYLENE GYANOL GREEN

ELECTROPHORESIS: GEL LOADING

AND DETECTION SYSTEMS

GEL LOADING
– Action of putting the isolated nucleic
acid in wells.
Prior to loading,
THINGS SHOULD BE ADDED:
• Tracking dyes
• Density agent

Tracking dyes – migrate in the gel and

allow visual monitoring or how fast
electrophoresis has proceeded.
– Migrate at specific speeds
in a given gel concentration.
– Usually run ahead of the
smallest fragments of DNA.
– They do not interfere to the
DNA separation.

ELECTROPHORESIS WILL TERMINATE

IF:

• DYE APPROACHES
TO THE END OF
THE GEL, OR
• OBTAINED THE
DESIRED
DISTANCE
TRACKING DYES
DETECTION SYSTEMS ▪ SYBR I – DSDNA -
EMIT @522nm
– Visualization of bands after ▪ SYBR II –
electrophoresis SSDNA/RNA – EMIT
@522nm
– MOST USED: ▪ SYBR GOLD –
• FLUORESCENT DYES DNA/RNA – EMIT
• SILVER STAIN @537nm

1. FLUORESCENT DYES 2. SILVER STAIN

INTERCALACTING AGENTS – ORIGINALLY DEVELOPED FOR
PROTEIN VISUALIZATION
– STACK (INTERCALATE) BETWEEN - Fixed with methanol
ADJACENT NITROGEN BASED IN + acetic acid
DOUBLE STRANDED NUCLEIC
ACIDS. - INSOLUBLE BLACK
SILVER AFTER
Example: Ethidium Bromide (EtBr) FORMALDEHYDE
• CARCINOGENIC
• EXCITATION @300 nm (UV)
• EMISSION @590nm
(ORANGE)
2 METHODS:
• BANDS WILL APPEAR AS
ORANGE IN COLOR. 1. SILVER
DIAMINE/AMMONIACAL
2 WAYS TO APPY:
SILVER
o Soaking in a solution of o BEST FOR
EtBr in running buffer THICK GELS
o Add directly before
polymerization/running 2. SILVER NITRATE
buffer o MOST STABLE
o BIOHAZARD
MINOR GROOVE-BINDING DYES
– SITS IN THE MINOR GROOVE OF
THE DOUBLE HELIX
Example: SYBR GREEN

• 25-100x MORE SENSITIVE

THAN EtBr
• PREFERRED FOR REAL-TIME
PCR
• EXCITES AT UV (300nm)
• SAFE TO USE