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Faheem Ahmad Roll No. 19
Hamayun Arshad Roll No. 07
Muhammad Ibraheem Roll No. 05

Class: M.Sc. 1st sem

Course code: 501

Madam Mamona

Institute of biochemistry and Biotechnology

University of Punjab Lahore
Introduction:
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to
amplify a single or a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA sequence.
Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable
technique used in medical and biological research labs for a variety of applications. These
include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of
genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used
in forensic sciences and paternity testing); and the detection and diagnosis of infectious
diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael
Smith for his work on PCR.
The method relies on thermal cycling, consisting of cycles of repeated heating and
cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers
(short DNA fragments) containing sequences complementary to the target region along
with a DNA polymerase (after which the method is named) are key components to enable
selective and repeated amplification. As PCR progresses, the DNA generated is itself
used as a template for replication, setting in motion a chain reaction in which the DNA
template is exponentially amplified. PCR can be extensively modified to perform a wide
array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq
polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This
DNA polymerase enzymatically assembles a new DNA strand from DNA building-
blocks, the nucleotides, by using single-stranded DNA as a template and DNA
oligonucleotides (also called DNA primers), which are required for initiation of DNA
synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating
and cooling the PCR sample to a defined series of temperature steps. These thermal
cycling steps are necessary first to physically separate the two strands in a DNA double
helix at a high temperature in a process called DNA melting. At a lower temperature,
each strand is then used as the template in DNA synthesis by the DNA polymerase to
 selectively amplify the target DNA. The selectivity of PCR results from the use of
primers that are complementary to the DNA region targeted for amplification under
specific thermal cycling conditions.

Requirements:
PCR amplification mix typically containing:
 Thermal cycler (thermocycler)
 Sample double stranded DNA with a target sequence
 Thermostable DNA polymerase e.g.,taq polymerase
 Two oligonucleotide primers
 Deoxynucleotide triphosphates (dNTPs)
 Reaction buffer containing magnesium ions in the form of MgCl2
 Sterile deionized water
 10X PCR buffer

Considerations:
Template DNA: Nearly any standard method is suitable for template DNA purification.
An adequate amount of template DNA is between 0.1 and 1 μg for genomic DNA for a
total reaction mixture of 100 μl. Larger template DNA amounts usually increase the yield
of non-specific PCR products.

Primers:
(1) PCR primers should be 10-24 nucleotides in length.
(2) The GC content should be 40%-60%.
(3) The primer should not be self-complementary or complementary to any other primer
in the reaction mixture, to prevent primer-dimmer and hairpin formation.
(4) Melting temperatures of primer pairs should not differ by more than 5°C, so that the
GC content and length must be chosen accordingly.
(5) The melting and annealing temperatures of a primer are estimated as follows: if the
primer is shorter than 25nucleotides, the approximate melting temperature is calculated
with the formula: Tm = 4(G + C) + 2 (A + T). (6) The annealing temperature should be
about 5°C lower than the melting temperature.
MgCl2 concentration: Because Mg2+ ions form complexes with dNTPs, primers and
 DNA templates, the optimal concentration of MgCl2 has to be selected for each
experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many will
increase the yield of non-specific products. The recommended range of MgCl2
concentration is 1 to 3 mM, under the standard reaction conditions specified.
Taq DNA polymerase:Higher Taq DNA polymerase concentrations than needed
maycause synthesis of non-specific products.
dNTPs: The concentration of each dNTP (dATP, dCTP, dGTP, dTTP) in the reaction
mixture is usually 200 μM. These concentrations must be checked as being equal, because
inaccuracies will increase the degree of disincorporation.

Procedure:
1. First of all we have to select a piece of DNA which has gene of interest. The DNA
molecule has two regions. One of which we have to amplify and carried our required
nucleotide sequence is known as target sequence. The other which is not to be amplify
is called flanking region

Figure 1: The DNA molecule that contain the gene of interest which is to be amplified (The target sequence).Before
starting the first the molecule is intact and containing hydrogen bonding. Flanking region is the region which is not to
be transcribed.

2. The DNA molecule carrying a target sequence is denatured by heat at 90-95oC for 20
seconds. The two strands separate due to breakage of the hydrogen bonds holding
them together. Oligonucleotide primers are then added.

Figure 2: At 95 0C the hydrogen bonding is broken and both strands of DNA move apart.
 3. A reaction mixture containing all four deoxynucleotide triphosphates (dATP, dCTP,
dGTP , dTTP) and a thermostable DNA polymerase is added. A DNA polymerase
(Taq) that is not denatured by the high temperature needed to separate the DNA
strands is used. It is usually sourced from Thermus aquaticus, abacterium isolated
from hot springs.

4. The mixture is allowed to cool to a lower temperature (50-65oC). Each strand of

DNA molecule becomes annealed with an oligonucleotide primer complementary to
either end of the target sequence. Primer annealing takes 20 seconds.

Figure 3: At 550C oligonucleotide primer is paired with the targeted DNA sequence

5. The temperature is raised to 60-75oC and primers are extended by the action of DNA
polymerase for 30seconds. The polymerase synthesizes complementary sequence the
5' to 3' direction away from each of the primers. If the template contains an A
nucleotide, the enzyme adds on a T nucleotide to the primer. If the template contains a
G, it adds a C to the new chain. Polymerization continues until each newly
synthesized strand has proceeded far enough to contain the site recognized by the
other primer. At this point there would be exactly two copies of the target DNA
sequence.

Figure 4: At 750Cthe targeted sequence has been replicated by the activity of Taq polymerase.
 6. The mixture is heated again at 90-95oC to denature the molecules and separate the
strands and the cycle repeated. Each new strand then acts as a template for the next
cycle of synthesis. Thus amplification proceeds at an exponential (logarithmic) rate,
i.e. amount of DNA produced doubles at each cycle. The amplified product at the end
of PCR is called amplicon.

typical thermal cycle might be as follows:

 Heat denaturation at 94oC for 60 second.
 Primer annealing at 54oC for 45 seconds.
 Primer extension at 72oC for 120 seconds
Average time for each cycle is approximately 4-5 minutes, considering the fact that
heating and cooling between each
stage also have to be considered.
Initially these steps at three
different temperatures were carried
out in separate water baths but
nowadays a thermocycler is used (a
machine that automatically changes
the temperature at the correct time for each of the stages and can be programmed to carry
out a set number of cycles). After the introduction of thermocycler, each cycle of
 replication can be completed in less than 5 minutes. After 30 cycles, a single molecule of
DNA is amplified into more than a billion copies (230 = 1.02 x 109).

Precautions:
 DNA extraction and PCR reaction mixing and processing should be performed in
separate areas.
 Use of sole-purpose vessels and positive displacement pipettes or tips for DNA
sample and reaction mixture preparation is strongly recommended.
 All solutions, except dNTPs, primers and Taq DNA polymerase, should be
autoclaved. Where possible, solutions should be aliquoted in small quantities and
stored in designated PCR areas.
 A good practice, to confirm absence of contamination, is to add a control reaction
without template DNA

Post amplification detection:

Following PCR, the amplification product can be detected using gel electrophoresis
followed by ethidium bromide staining and visualization with UV trans illumination.
Visualization of a band containing DNA fragment of a particular size can indicate the
presence of the target sequence in the original DNA sample. Absence of a band may
indicate that the target sequence was not present in the original DNA sample.
Confirmation of the amplicons can be made by southern blotting using specific probes.

Applications of PCR:
The polymerase chain reaction is used by a wide spectrum of scientists in an ever-
increasing range of scientific disciplines. In microbiology and molecular biology, for
example, PCR is used in research laboratories in DNA cloning procedures, Southern
blotting, DNA sequencing, recombinant DNA technology, to name but a few. In clinical
microbiology laboratories PCR is invaluable for the diagnosis of microbial infections and
epidemiological studies. PCR is also used in forensics laboratories and is especially
useful because only a tiny amount of original DNA is required, for example, sufficient
DNA can be obtained from a droplet of blood or a single hair.
PCR has wide application in biochemistry.A brief summary is following:
 Amplification of small amounts of DNA for further analysis by DNA fingerprinting.
 The analysis of ancient DNA from fossils.
  Mapping the human (and other species) genome.
 The isolation of a particular gene of interest from a tissue sample.
 Generation of probes: large amount of probes can be synthesized by this technique.
 Production of DNA for sequencing: Target DNA in clone is amplified using
appropriate primers and then its sequence determined. It is helpful in conditions
where amount of DNA is small.
 Analysis of mutations: Deletions and insertions in a gene can be detected by
differences in size of amplified product.
 Diagnosis of monogenic diseases (single gene disorders): For pre-natal diagnosis,
PCR is used to amplify DNA from fetal cells obtained from amniotic fluid. PCR has
also proved very important in carrier testing.
 Detection of microorganisms: Especially of organisms and viruses that are difficult to
culture or take longtime to culture or dangerous to culture.
 The PCR has even made it possible to analyze DNA from microscope slides of tissue
preserved years before.
 Detection of microbial genes responsible for some aspect of pathogenesis or antibiotic
resistance.
 Crucial forensic evidence may often be present in very small quantities, e.g. one
human hair, body fluid stain (blood, saliva, semen). PCR can generate sufficient DNA
from a single cell.

Limitations of PCR:
PCR is an extremely sensitive technique but is prone to contamination from
extraneous DNA, leading to false positive results. Another potential problem is due to
cross-contamination between samples. It is for this reason that sample preparation,
running PCR and post-amplification detection must be carried out in separate rooms.
Concentration of Mg is very crucial as low Mg2+ leads to low yields (or no yield) and
high Mg2+ leads to accumulation of nonspecific products. Non-specific binding of
primers and primer-primer dimmer formation are other possible reasons for unexpected
results. Reagents and equipment are costly, hence can’t be afforded by small laboratories.

Types of PCR:
1. Amplified fragment length polymorphism PCR (AFLP PCR)
2. Allele-specific PCR
3. ALU-PCR
4. Assembly PCR
5. Asymmetric PCR
6. Colony PCR.
7. Helicase dependent amplification.
8. Hot start PCR.
9. Inverse PCR.
10. In situ PCR.
11. ISSR -PCR.
12. RT-PCR(REVERSE TARNSCRIPTASE)
13. REAL TIME PCR.
14. Nested PCR
15. Multiplex PCR

1). Amplified fragment length polymorphism PCR (AFLP PCR)

Amplified Fragment Length Polymorphism PCR, also called AFLP PCR was originally
described by Zabeau in1993.Developed in the early 1990s by Keygene,AFLP uses
restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky
ends of the restriction fragments. A subset of the restriction fragments is then selected to
be amplified. This selection is achieved by using primers complementary to the adaptor
sequence, the restriction site sequence and a few nucleotides inside the restriction site
fragments.The amplified fragments are visualized on denaturing polyacrylamide gels
either through autoradiography or fluorescence methodologies. It is a highly sensitive
method for detecting polymorphisms in DNA

AFLP is composed of 3 steps:

1-A) Cellular DNA is digested with one or more restriction enzymes.
1-B) Ligation of linkers (restriction half-site specific adaptors) to all restriction fragments.
2) Pre-selective PCR is performed using primers which match the linkers and restriction
site specific sequences.
 3) Electrophoretic separation and amplicons on a gel matrix, followed by visualization of
the band pattern.

Applications of AFLP PCR

AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA.It is

a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of
genetic engineering.

2). Allele-specific PCR

Allele-specific PCR is a varation of the polymerase chain reaction which is used as a
diagnostic or cloning technique, to identify or utilize single-nucleotide polymorphisms
(SNPs). Allele-specific PCR does require the sequence of the target DNA sequence,
including differences between the alleles.It is a convenient and inexpensive method for
genotyping Single Nucleotide Polymorphisms (SNPs) and mutations. It is applied in many
recent studies including population genetics, molecular genetics and
pharmacogenomics.

Primers for Allele-Specific PCR

The allele-specific PCR uses primers whose 3' ends encompass the SNP.
PCR amplification under stringent conditions is much less efficient in the presence of a
mismatch between template and primer, so successful amplification with a SNP-specific
primer signals presence of the specific SNP in a sequence.

3). ALU PCR

PCR using a primer that anneals to Alu repeats to amplify DNA located between two
oppositely oriented Alu sequences. Used as a method of obtaining a fingerprint of bands
from an uncharacterized human DNA.

4). Assembly –PCR

Assembly PCR can be used to assemble two gene-sized pieces of DNA into one piece for
easier cloning of fusion genes/parts. Briefly, it essentially involves PCR'ing the two
pieces separately with primers that have a 20bp overlap and then doing an extra PCR step
using the two products as the template. This is essentially just for ease of cloning. Instead
of trying to PCR or cut out of a vector two separate pieces and then assemble them by
 endonuclease digestion and ligation (aka 3-way ligation), it can be easier simply to PCR
the first piece w/ a reverse primer that overlaps with the forward primer of the second
piece, and then use the product of the first PCR reactions as a template for the assembly
reaction. If the reverse primer for the 5' piece and the forward primer for the 3' piece
overlap by ~20bp, the product of the first PCR reactions should anneal in the overlapping
region and create a full length (gene fusion) product. Using the forward primer for the
first piece and the reverse primer for the second piece in the assembly reaction then
amplifies the desired full-length product. The merits for this technique are that it's
arguably faster than standard 3-way ligation assembly (because you need good-quality
DNA to make that work well, which usually means sub-cloning each piece), and it's more
reliable (the quality of the product is very good so you can clone it directly into the
desired vector.
5). Asymmetric –PCR
Asymmetric PCR is used to preferentially amplify one strand of the target DNA more
than the other. A PCR in which the predominant product is a single-stranded DNA as a
result of unequal primer concentrations. As asymmetric PCR proceeds, the lower
concentration primer is quantitatively incorporated into double-stranded DNA. The
higher concentration primer continues to primer synthesis, but only of its strand.

Applications of Asymmetric PCR

The Asymmetric PCR is useful in some sequencing and hybridization probing
applications where having only one of the two complementary stands is sufficient or
required.

6). Colony –PCR

Colony PCR the screening of bacterial (E. coli) or yeast clones for correct ligation or
plasmid products. Selected colonies of bacteria or yeast are picked with a pipette tip from
a growth (agarose) plate. This is then inserted into the PCR master mix or pre-inserted
into autoclaved water. PCR is then conducted to determine if the colony contains the
DNA fragment or plasmid of interest.
It can be used after a transformation to screen colonies for the desired plasmid. Primers
are used which generate a PCR product of known size. Thus, any colonies which give rise
to an amplification product of the expected size are likely to contain the correct DNA
sequence.
7). Helicase dependent amplification (HDA)
The polymerase chain reaction is the most widely used method for in vitro DNA
amplification for purposes of molecular biology and biomedical research. This process
involves the separation of the double-stranded DNA in high heat into single strands (the
denaturation step, typically achieved at 95–97 °C), annealing of the primers to the single
stranded DNA (the annealing step) and copying the single strands to create new double-
stranded DNA (the extension step that requires the DNA polymerase) requires the
reaction to be done in a thermal cycler. In vivo, DNA is replicated by DNA polymerases
with various accessory proteins, including a DNA helicase that acts to separate the DNA
by unwinding the DNA double helix.HDA was developed from this concept, using a
helicase (an enzyme) to denature the DNA.

8). Hot start PCR

Hot Start PCR allows the inhibition of polymerase activity during PCR reaction
preparation. By limiting polymerase activity prior to PCR cycling.Hot Start PCR reduces
non-specific amplification and increases PCR product target yield. It is commonly
performed by using included chemical modifications, wax-barrier methods, and inhibition
by a taq-directed antibody.
PCR or polymerase chain reaction uses DNA polymerases which were isolated from
thermostable organisms such as the Taq enzyme and Pfu.
PCR amplifies DNA by multiple cycles of melting DNA at high temperature, annealing
primers at a lower temperature, and then allowing polymerase extension at an
intermediate temperature.
This in-efficient DNA polymerase activity will catalyze the extension of any annealed 3'
ends. After PCR amplification, the resulting product contains a mixture of specific and
non-specific bands. Moreover, the 5'-3' exonuclease activity of the DNA polymerase l
degrades any free 5' ends of partially annealed nucleic acids, destroying the primer and
template substrates of the polymerase reaction. This results in a lower yield of the desired
pcr product.

9) Inverse –PCR (IPCR)

Inverse PCR also called IPCR, and was first described by Ochman et al. in 1988. A
limitation of standard PCR is that 5' and 3' flanking regions of your DNA fragment of
 interest must be known. Inverse PCR allows you to conduct PCR when you only have the
information of one internal sequence.

Inverse polymerase chain reaction is a variant of PCR, and is used when only one internal
sequence of the target DNA is known. It is therefore very useful in identifying flanking
DNA sequences of genomic inserts. Similar to other PCR methods, inverse PCR
amplifies target DNA using DNA polymerase.

The Inverse PCR Method

The inverse PCR method includes a series of digestions and self-ligations with the DNA
being cut by a restriction endonuclease. This cut results in a known sequence at either end
of unknown sequences.

Applications of Inverse PCR

Inverse PCR has numerous applications in molecular biology including the amplification
and identification of sequences flanking transposable elements and the identification of
genomic inserts.

10). Insitu –PCR

In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell
on a slide. In situ PCR amplification can be performed on fixed tissue or cells.

11). ISSR-PCR
Inter-simple sequence repeat (ISSR) PCR is a fast and inexpensive genotyping technique
with a wide range of uses, including the characterization of genetic relatedness among
populations. Despite its utility, the ISSR-PCR method has typically suffered from poor
reproducibility and time-consuming data analysis.

ISSR-PCR is a genotyping technique based on variation found inthe regions between

microsatellites. It has been used in genetic fingerprinting, gene tagging, detection of
clonal variation cultivar identification, phylogenetic analysis, detection of genomic
instability and assessment of hybridization in many plant and animal species. The
versatility of this genotyping technique makes ISSR useful for researchers interested in
diverse fields such as conservation biology and cancer research.
12). Reverse Transcriptase PCR
Reverse transcription polymerase chain reaction (RT-PCR) is based on the polymerase
chain reaction (PCR). More importantly it is based on the process of reverse transcription,
which reverse transcribes RNA into DNA and was initially isolated from retroviruses.
The techniques of RT-PCR allows the formation of cDNA (complementary or copy DNA)
from RNA, which stores the sequence of RNA (such as messenger RNA, mRNA) in the
more stable form of nucleic acid, DNA. This reverse transcription from RNA into its
reverse complement DNA (cDNA) is the first step of a usually two-step process of RT-
PCR. Furthermore, by copying the RNA into DNA, one can then amplify the c DNA
sequence by using primers specific for the DNA sequence. This amplification is the final
second major step of the two-step process of RT-PCR.

13) Nested PCR

Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs
(instead of one pair) of PCR primers are used to amplify a fragment.

The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a
second pair of primers called nested fragment bind inside the first PCR product fragment
to allow amplification of a second PCR product which is shorter than the first one.

14). Multiplex PCR

Multiplex polymerase chain reaction (Multiplex PCR) is a modification of polymerase
chain reaction in order to rapidly detect deletions or duplications in a large gene. This
process amplifies genomic DNA samples using multiple primers and a temperature-
mediated DNA polymerase in a thermal cycler. Multiplex-PCR was first described in
1988 as a method to detect deletions in the dystrophin gene.It has also been used with the
steroid sulfatase gene.In 2008, multiplex-PCR was used for analysis of microsatellites
and SNPs.

Applications
Some of the applications of multiplex PCR include:
1. Pathogen Identification
2. High Throughput SNP Genotyping
3. Mutation Analysis
 4. Gene Deletion Analysis
5. Template Quantitation
6. Linkage Analysis
7. RNA Detection
8. Forensic Studies

Real-Time PCR The latest type

An Introduction to Real-Time PCR

The development of instruments that allowed real-time monitoring of fluorescence within
PCR reaction vessels was a significant advance. The technology is flexible and many
alternative instruments and fluorescent probe systems have been developed and are
currently available. Real-time PCR assays can be completed rapidly since no
manipulations are required post-amplification. Identification of the amplification products
by probe detection in real-time is highly accurate compared with size analysis on gels.
Analysis of the progress of the reaction allows accurate quantification of the target
sequence over a very wide dynamic range, provided suitable standards are available.
Further investigation of the real-time PCR products within the original reaction mixture
using probes and melting analysis can detect sequence variants including single base
mutations. Since the first practical demonstration of the concept real-time PCR has found
applications in many branches of biological science. Applications include gene
expression analysis, the diagnosis of infectious disease and human genetic testing. Due to
their fluorimetry capabilities, these real-time machines are also compatible with
alternative amplification methods such as NASBA, provided a fluorescence end-point is
available.

An Overview of PCR Platforms

Real-time PCR continues to have a major impact across many disciplines of the biological
sciences and this has been a driver to develop
and improve existing instruments. From the
first two commercial platforms introduced in
the mid-1990s, there is now a wide choice of
instruments, which continues to increase.
Advances include faster thermocycling times,
higher throughput, flexibility, expanded optical systems, increased multiplexing and more
user-friendly software. The main features of each instrument are compared and factors
important to weigh up when deciding on a platform are highlighted.

Homogeneous Fluorescent Chemistries for Real-Time PCR

The development of fluorescent methods for a closed tube polymerase chain reaction has
greatly simplified the process of nucleic acid quantification. Current approaches use
fluorescent probes that interact with the amplification products during the PCR allowing
kinetic measurement of product accumulation. These probe methods include generic
approaches to DNA quantification such as fluorescent DNA binding dyes. There are also
a number of strand-specific probes that use the phenomenon of Fluorescent Energy
Transfer. In this chapter we describe these methods in detail, outline the principles of
each process, and describe published examples. This text has been written to provide an
impartial overview of the utility of different assays and to show how they may be used on
various commercially available thermal cyclers.

Reference Gene Validation Software for Improved Normalization

Real-time PCR is the method of choice for expression analysis of a limited number of
genes. The measured gene expression variation between subjects is the sum of the true
biological variation and several confounding factors resulting in non-specific variation.
The purpose of normalization is to remove the non-biological variation as much as
possible. Several normalization strategies have been proposed, but the use of one or more
reference genes is currently the preferred way of normalization. While these reference
genes constitute the best possible normalizers, a major problem is that these genes have
no constant expression under all experimental conditions. The experimenter therefore
needs to carefully assess whether a certain reference gene is stably expressed in the
experimental system under study. This is not trivial and represents a circular problem.
Fortunately, several algorithms and freely available software have been developed to
address this problem. This chapter aims to provide an overview of the different concepts.
Data Analysis Software

Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of
mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic
quantification range. While qRT-PCR has a tremendous potential for analytical and
quantitative applications, a comprehensive understanding of its underlying principles is
important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and
polymerase performances, the fidelity of the quantification process is highly dependent on a
valid data analysis. This review will cover all aspects of data acquisition (trueness,
reproducibility, and robustness), potentials in data modification and will focus particularly on
relative quantification methods. Furthermore useful bioinformatical, biostatical as well as
multi-dimensional expression software tools will be presented.

Performing Real-time PCR

Optimisation of the reagents used to perform PCR is critical for reliable and reproducible
results. As with any PCR initial time spent on optimisation of a real-time assay will be
beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the
important criteria to consider when optimising an assay and these can be affected by changes
in the primer concentration, probe concentration, cycling conditions and buffer composition.
An optimised real-time PCR assay will display no test-to-test variation in the crossing
threshold or crossing point and only minimal variation in the amount of fluorescence. The
analysis of the real-time PCR results is also an important consideration and this differs from
the analysis of conventional block-based thermal cycling. Real-time PCR provides
information on the cycle at which amplification occurs and on some platforms the melting
temperature of the amplicon or probe can be determined.

Internal and External Controls for Reagent Validation

False negatives in PCR can occur from inhibition of one or more of the reaction components
by a range of factors. Therefore applications requiring a high level of confidence in the result
need to be designed to control for the occurrence of false negatives. While an external, or
batch, control is often used, the ideal control is one that is included in the reaction cocktail in
a multiplex format. Here we discuss the application and development of molecular mimics
for use as controls in real-time PCR, and explain a number of concepts and experimental
considerations that will aid in the optimisation of controlled multiplexed assays.

Introduction to the Applications of Real-Time PCR

The technique of real-time PCR has features that make its use advantageous in a wide range
of applications. A number of examples, covering the main areas of application, are given in
the following chapters of this book. In this introduction the important features of these
applications are discussed.

Analysis of mRNA Expression by Real-Time PCR

Its conceptual and practical simplicity, capacity for high throughput, and combination of
high sensitivity with exacting specificity has made the fluorescence-based real-time reverse
transcription polymerase chain reaction (qRT-PCR or RT-qPCR) today's method of choice
for the quantification of RNA. The technology continues to evolve rapidly with the
introduction of new protocols, enzymes, chemistries and instrumentation and has become the
"Gold Standard" for a huge range of applications in basic research, molecular medicine, and
biotechnology. Progress is increasingly associated with an increased appreciation of the
limitations associated with this technology and the need for careful experimental design,
application and validation.

Validation of Array Data

Microarray techniques allow the parallel assessment of the relative expression of thousands
of transcripts in response to different experimental conditions or in different tissues. The
ability to correctly identify differentially expressed genes is limited by the signal to noise
ratio, the variation in the levels of gene expression, and/or the variability in the measurements
due to the assay itself. Therefore, an unequivocal identification of differentially expressed
transcripts requires independent confirmation. Quantitative real-time RT-PCR (qPCR) is the
method of choice because of its broad range of linearity. Furthermore, it can be easily
adapted to systematically study tens to hundreds of different transcripts. The cDNA
microarray technique is introduced as an example, followed by comparisons to different
microarray platforms and their characteristics. Data analysis of microarray experiments will
show the importance of verification of results. General differences between microarray
hybridisations and PCR reactions and, in particular, the performance of different platforms
are described and compared. Furthermore, the effects of increasing tissue complexity on
detection of differentially expressed transcripts are elucidated with specific examples.

Mutation Detection by Real-Time PCR

Real-time applications for mutation detection include detecting alterations associated with
inherited disease, acquired alterations in oncology, and microbial or viral mutations
associated with drug resistance in infectious diseases. Probe chemistries described for these
applications include hydrolysis (TaqMan®) and hybridisation probes (FRET and Molecular
Beacons). Hydrolysis probes detect mutations by allele specific hybridisation at a specific
temperature, while hybridisation probes allow dynamic detection through a temperature
range. Primer chemistries are described for allele specific amplification and Scorpion
primers. Recent progress in scanning amplicons for mutations also includes high resolution
melting. The design of each of these methods is described, along with applications.

Real-Time NASBA

NASBA is an isothermal nucleic acid amplification method which is particularly suited to

detection and quantification of genomic, ribosomal or messenger RNA. The product of
NASBA is single-stranded RNA of opposite sense to the original target. First developed
NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification
detection of products. More recently, real-time procedures incorporating amplification and
detection in a single step have been reported and applied to a wide range of RNA and some
DNA targets. Thus real-time NASBA has proved to be the basis of sensitive and specific
assays for detection, quantification and differentiation of RNA and DNA targets. Molecular
beacons have most often been utilised in real-time NASBA whether in commercially-
available kits or as published in-house developed assays. As experience in design of
molecular-beacon probes increases and fluorimeters suitable for real-time NASBA become
widely available this methodology will be confirmed as a suitable alternative to real-time RT-
PCR (and perhaps DNA PCR).

Applications in Clinical Microbiology

The introduction of real-time PCR assays to the clinical microbiology laboratory has led to
significant improvements in the diagnosis of infectious disease. There has been an explosion
of interest in this technique since its introduction and several hundred reports have been
published describing applications in clinical bacteriology, parasitology and virology. There
are few areas of clinical microbiology which remain unaffected by this new method. It has
been particularly useful to detect slow growing or difficult to grow infectious agents.
However, its greatest impact is probably its use for the quantitation of target organisms in
samples. The ability to monitor the PCR reaction in real-time allows accurate quantitation of
target sequence over at least six orders of magnitude. The closed-tube format which removes
the need for post-amplification manipulation of the PCR products also reduces the likelihood
of amplicon carryover to subsequent reactions reducing the risk of false-positives. As more
laboratories begin to utilise these methods standardisation of assay protocols for use in
diagnostic clinical microbiology is needed, plus participation in external quality control
schemes is required to ensure quality of testing.

Diagnosis of Invasive Fungal Infections

The mounting prevalence of invasive fungal disease in immunocompromised patients is

exacerbated by inadequate methods for pathogen detection. PCR-based amplification
approaches have been developed to address this problem because conventional methods for
pathogen identification lack sensitivity, specificity and speed, and some infectious organisms
are difficult to culture. PCR amplification of ribosomal genes and their internal transcribed
spacer regions coupled with sequence-specific detection probes are the most reliable
approaches for fungal identification. Real-time self-reporting probes capable of single
nucleotide allelic discrimination have expanded PCR applications to target mechanisms of
drug resistance. Clinical applications of PCR are expanding for diagnosing invasive fungal
diseases in blood and respiratory specimens at an early stage to improve treatment outcomes
for high risk patients.

Biodefense
With the public's reawakened concern regarding use of biological agents as weapons, the
rapid detection, discrimination, and identification of pathogenic organisms and toxins has
become a priority for state and federal government agencies. High confidence, cost effective,
and near real-time diagnostic methods are essential to protecting national health security
whether the target is public health, agriculture, commodities, or water supply infrastructures.
While culture-based methods have been, and will likely remain, the gold standard for
microbiological diagnostics, PCR-based tests offer significant advantages in sensitivity,
specificity, speed and data richness that make them invaluable to diagnostic laboratories. In
this chapter, we will describe the application of real-time PCR methods in biodefense. We
will discuss the use of real-time PCR in biodefense in terms of general workflow and
processing considerations, clinical diagnostic applications, environmental diagnostic
applications, and multiplex screening. Real-time PCR assays can be either quantitative
(qPCR) or qualitative, depending on whether a standard curve is included with the analytical
run. Most diagnostic and biodefense applications utilise the qualitative nature of real-time
PCR as a detection platform; this chapter will focus on the benefits of these types of assays.
Finally, we will consider the future uses and anticipated advances in real-time PCR
applications as related to biodefense.

Real-Time PCR: Application to Food Authenticity and Legislation

Real-time PCR is now an accepted analytical tool within the food industry. Its principal
role has been one of assisting the legislative authorities, major manufacturers and retailers
to confirm the authenticity of foods. The most obvious role is the detection and
quantification of GMOs, but real-time PCR makes a significant contribution to many
other areas of the food industry, including food safety and other speciality analyses such
as the detection of common wheat adulteration in pasta and the detection of allergenic
species. The role of quantitative real-time PCR in determining the actual amount of these
materials, which are subject to considerable regulation, is discussed together with a
consideration of the uncertainty of the methods.

Molecular Haplotyping by Real-time PCR

Molecular real-time PCR methods can determine whether two or more mutations are on
the same or different chromosomes. This ability to haplotype without family studies is
useful for research and clinical purposes and can give an advantage over genotyping.
Haplotyping by real-time PCR with hybridization probes has been demonstrated for
adjacent repeats and single base alterations, with a probe that covers both sites. However,
base alterations may be separated by distances greater than a traditional hybridisation
probe will cover. We described a probe design that covers both (or all) sites, but does not
include the entire sequence between the sites. When hybridized with the template, the
template is forced to form a loop. This "loop-out" probe will dissociate from the template
as a unit, therefore allowing haplotyping of base alterations separated by over 80 bp.
Examples of haplotyping by traditional probes for adjacent sequence variants, as well as
examples of "loop-out" probes are presented.

References

1) Real time PCR

Logan J, Edwards K. Real-Time PCR: Current Technology and Applications, 2009.Caister

Academic Press. Review is available at http://www.horizonpress.com/realtimepcr

2) PCR Principle
http://users.ugent.be/~avierstr/principles/pcr.html
3) Principle and consideration
http://www2.bioversityinternational.org/Publications/Molecular_Markers_Volume_1_en/
molmarkers/PDF/PCR%20basics.pdf
4) Higuchi R, Dollinger G, Walsh PS, Griffith R: Simultaneous amplification and detection of
specific DNA sequences. Bio/Technology 10: 413–417, 1992
5) Wilfingseder D, Stoiber H: Quantifizierung von PCR-Produktmengen durch real-time
PCRVerfahren.
Antibiotika Monitor, Heft 1/2/2002
6) Reidhaar-Olson JF, Hammer J: The impact of genomics tools on target discovery. Curr Drug
 Discovery, April 2001
7) Brock TD: Life at high temperatures. Bacteriology 303: Procaryotic Microbiology, 1994
8) http://www.bact.wisc.edu/Bact303/b27
9) Mullis KB: The polymerase chain reaction. Nobel Lecture, December 8, 1993
10) http://www.nobel.se/chemistry/laureates/1993/mullis-lecture.html
11) Kary Mullis Website: http://www.karymullis.com
12) Deutsches Hepatitis C Forum e.V. Homepage – Qualitativer HCV-RNA Nachweis: