You are on page 1of 7


A polymerase chain reaction is a method of amplifying a certain region of DNA sequence. It is an in vitro
technique that is used to generate large quantities of a specified DNA even from the smallest quantities of
available DNA like a drop of blood or a single strand of hair. A polymerase chain reaction has different types which
have been developed to improve the conventional PCR and/or cater to the needs of certain DNA sequences.
Real-time PCR is an advanced form of the Polymerase Chain Reaction that maximizes the potential of the
technique. The conventional PCR detects the the amplified product, or amplicon, by an end-point analysis by
running DNA on an agarose gel after the reaction has finished. Real-time PCR on the other hand, allows the
accumulation of amplified product to be detected and measured as the reaction progresses, that is, in real time.

This method detects PCR products by including a fluorescent molecule in the reaction that reports an increase in
DNA count. The fluorescent signal is directly proportional to the amount of DNA. The fluorescent chemistries used
has DNA-binding dyes and fluorescently labeled sequence-specific primers or probes. Specialized thermal cyclers
equipped with fluorescence detection modules are used to monitor the fluorescence as amplification occurs. The
measured fluorescence reflects the amount of amplified product in each cycle.
The main advantage of real-time PCR over conventional PCR is that real-time PCR allows you to determine the
starting template copy number with accuracy and high sensitivity over a wide dynamic range. Real-time PCR
results can either be qualitative (presence or absence of a sequence) or quantitative (number of copies of DNA).
Real-time PCR that is quantitative is also known as qPCR. In contrast, conventional PCR is at best semiquantitative. Additionally, real-time PCR data can be evaluated without gel electrophoresis, resulting in reduced
experiment time and increased throughput. Finally, because reactions are run and data are evaluated in a closedtube system, opportunities for contamination are reduced and the need for post-amplification manipulation is
Real time PCR has many benefits compared to the conventional approach:

It gives an observer a look in to the reaction. You can literally see which reactions have worked well and

which have failed.

The efficiency of the reaction can be precisely calculated.
There is also no need to run the PCR product out on a gel after the reaction as the melt curve analysis

effectively does this for you.

The greatest advantage of all however, is that real-time PCR data can be used to perform truly
quantitative analysis of gene expression. In comparison, old fashioned PCR was only ever semiquantitative at best.

Multiplex polymerase chain reaction (PCR) is a type of PCR that has two or more target sequences that can be
amplified by including more than one pair of primers in the same reaction. Multiplex PCR has the potential to
produce considerable savings of time and effort in the laboratory. Since it was first described in 1988, this method
has been successfully applied in many areas of DNA testing, including gene deletion analysis, mutation and
polymorphism analysis, quantitative analysis, and reverse-transcription (RT)-PCR. In the field of infectious
diseases, multiplex PCR has been shown to be a valuable tool for identification of viruses, bacteria, and parasites.
Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR
experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer
pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to
produce considerable savings in time and effort within the laboratory without compromising on the utility of the
Types of Multiplex PCR
Multiplexing reactions can be broadly divided in two categories:
1. Single Template PCR Reaction
This technique uses a single template which can be a genomic DNA along with several pairs of forward and
reverse primers to amplify specific regions within a template.
2. Multiple Template PCR Reaction
It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may
lead to cross hybridization with each other and the possibility of mis-priming with other templates.
Primer Design Parameters for Multiplex PCR
Design of specific primer sets is essential for a successful multiplex reaction. The important primer design
considerations described below are a key to specific amplification with high yield.
1. Primer Length
Multiplex PCR assays involve designing of large number of primers, hence it is required that the designed primer
should be of appropriate length. Usually, primers of short length, in the range of 18-22 bases are used.
2. Melting Temperature
Primers with similar Tm, preferably between 55C-60C are used. For sequences with high GC content, primers
with a higher Tm (preferably 75C-80C) are recommended. A Tm variation of between 3-5 C is acceptable for
primers used in a pool.

3. Specificity
It is important to consider the specificity of designed primers to the target sequences, while preparing a multiplex
assay, especially since competition exists when multiple target sequences are in a single reaction vessel.
4. Avoid Primer Dimer Formation
The designed primers should be checked for formation of primer dimers, with all the primers present in the
reaction mixture. Dimerization leads to unspecific amplification.
All other parameters are similar to standard PCR primer design guidelines.
Advantages of Multiplex PCR
1. Internal Controls
Potential problems in a simple PCR include false negatives due to reaction failure or false positives due to
contamination. False negatives are often revealed in multiplex assays because each amplicon provides an
internal control for the other amplified fragments.
2. Efficiency
The expense of reagents and preparation time is less in multiplex PCR than in systems where several tubes of
uniplex PCRs are used. A multiplex reaction is ideal for conserving costly polymerase and templates in short
3. Indication of Template Quality
The quality of the template may be determined more effectively in multiplex than in a simple PCR reaction.
4. Indication of Template Quantity
The exponential amplification and internal standards of multiplex PCR can be used to assess the amount of a
particular template in a sample. To quantitate templates accurately by multiplex PCR, the amount of reference
template, the number of reaction cycles, and the minimum inhibition of the theoretical doubling of product for each
cycle must be accounted.
Applications of Multiplex PCR

Pathogen Identification
High Throughput SNP Genotyping
Mutation Analysis
Gene Deletion Analysis
Template Quantitation
Linkage Analysis
RNA Detection
Forensic Studies

TD-PCR is an innovation of PCR in which the initial annealing temperature is higher than the optimal Tm of the
primers and is gradually reduced over subsequent cycles until the Tm temperature or touchdown temperature is
reached, much like the touchdown of an airplane.
A gradual lowering of temperature to a more permissive annealing temperature during the course of cycling favors
amplification of the desired amplicon.
Why does TD-PCR work better?
Optimal annealing temperature is a requirement in PCR. This is normally determined based on the melting
temperature (Tm) of the primer-template pair. But, primer Tm is affected variously by the individual buffer
components, even primer and template concentrations so any calculated primer Tm value is only an
approximation. Therefore, it is often difficult to find the right annealing temperature for a given primer/template
Too-low annealing temperatures, can lead to primer-dimer formation and non-specific products while too-high
temperatures reduce yield due to poor primer annealing.
By using temperatures higher than the calculated T m in the initial cycles, TD-PCR favors only accumulation of
amplicons whose primer-template complementarity is the highest. The stepwise transition to a lower temperature
during subsequent cycle guards against lower yields by making use of the desired amplicons in the reaction that
now outcompetes any non-specific products or primer-dimers if present.
TD-PCR cycling conditions
The protocol published in Nature Protocols works very well and is a good reference to start off with TD-PCR.
The suggested cycling program has two phases. The first phase of touchdown programming uses a Tm that is
approximately 10C above the calculated Tm. The temperature is reduced by 1C every successive cycle until the
calculated Tm range is reached. This is done for a total of 10-15 cycles.
Phase 2 follows generic PCR amplification of up to 20-25 cycles using the final annealing temperature reached in
the touchdown phase.
The cycles and temperature drop during touchdown phase can be adjusted from 1-3 cycles per 1-3C drop in
temperature if non-specific products are still observed or if the yield is low.

Assembly PCR, using synthetically derived DNA, is a very flexible technique for producing novel gene sequences.
Single-stranded oligos or a mix of single- and double-stranded DNA are used to produce longer genes of up to
several thousand base pairs. The approach can also be beneficial for assembling constructs with modular
elements, such as antibodies. Assembly PCR is also interesting because overlapping sequences can be joined
without the need for restriction sites, and one can take advantage of robust PCR reagents and methods. However,
because assembly PCR usually involves putting together many short fragments, experiments require careful
planning and substantial optimization to be successful.
Overall flexibility and the low cost of standard oligonucleotides make assembly PCR seem like an easy choice for
gene construction. However, there are several considerations that make the technique, in practice, more
challenging. In addition to design and logistics factors, the success of assembly PCR is affected by the same
factors that affect regular PCR.
Annealing temperatures. Overlapping sequences should have annealing temperatures (Tm) ideally between 60
and 70C and within 5C for all termini of the DNA elements being assembled.
Oligonucleotide characteristics. GC content, secondary structure, and repetitive sequences can affect annealing,
amplification, and cloning so some sequence optimization may be necessary for successful assembly. In many
cases this can be accomplished following existing knowledge and guidelines for PCR.
PCR conditions.
Reaction conditions can be optimized for assembly PCR. Adjusting DNA, dNTP, Mg2+, and enzyme
concentrations may be helpful, and inhibitors of PCR, such as chelators and organic solvents, should be avoided.
Primer concentration
Commonly, the outermost primers in an assembly PCR are at higher concentrations, approximately 30 pmol, for
amplification of the overall construct, and the internal primers or double-stranded DNA is kept at lower
concentrations, approximately 1.52 pmol (Figure 1) [1].
Sequence errors.
Another concern with assembly PCR is that a subpopulation of the synthetic oligonucleotides contains small
errors that arise during synthesis. The low rate of these errors in quality oligonucleotides is typically not an issue
for PCR amplifications because the vast majority of amplified products will be correct. However, for assembly
PCR, the statistical probability of one or more of these errors showing up in the final sequence increases with the
number of oligos assembled, as well as the lengths of oligos used.

High-fidelity PCR, or low error rate, is a type of PCR that uses proofreading enzymes with 3 to 5 exonuclease
activity, such as Platinum Taq High Fidelity. High-fidelity PCR, utilizes a DNA polymerase with a low error rate
and results in a high degree of accuracy in the replication of the DNA of interest. It is required in applications
where sequence accuracy is crucial.

Examples include cloning, genetic profiling, and next-generation

sequencing. Routine high-fidelity amplification of longer and difficult targets with wild-type proofreading
polymerases is challenging due to the low processivity and lack of robustness of these enzymes.
The fidelity of a polymerase refers to its ability to insert the correct base during PCR. Conversely, the rate of
misincorporation is known as a polymerases error rate.
What is fidelity?
The fidelity of a DNA polymerase is the result of accurate replication of a desired template. Specifically, this
involves multiple steps, including the ability to read a template strand, select the appropriate nucleoside
triphosphate and insert the correct nucleotide at the 3 primer terminus, such that Watson-Crick base pairing is
maintained. In addition to effective discrimination of correct versus incorrect nucleotide incorporation, some DNA
polymerases possess a 35 exonuclease activity. This activity, known as proofreading, is used to excise
incorrectly incorporated mononucleotides that are then replaced with the correct nucleotide. High-fidelity PCR
utilizes DNA polymerases that couple low misincorporation rates with proofreading activity to give faithful
replication of the target DNA of interest.
When is fidelity important?
Fidelity is important for applications in which the DNA sequence must be correct after amplification. Common
examples include cloning/subcloning DNA for protein expression, SNP analysis and next generation sequencing
applications. Fidelity is less important for many diagnostic applications where the read-out is simply the presence
or absence of a product.
How does a high-fidelity polymerase ensure that the correct base is inserted?
High-fidelity DNA polymerases have several safeguards to protect against both making and propagating mistakes
while copying DNA. Such enzymes have a significant binding preference for the correct versus the incorrect
nucleoside triphosphate during polymerization. If an incorrect nucleotide does bind in the polymerase active site,
incorporation is slowed due to the sub-optimal architecture of the active site complex. This lag time increases the
opportunity for the incorrect nucleotide to dissociate before polymerase progression, thereby allowing the process
to start again, with a correct nucleoside triphosphate (1,2). If an incorrect nucleotide is inserted, proofreading DNA
polymerases have an extra line of defense (Figure 1). The perturbation caused by the mispaired bases is
detected, and the polymerase moves the 3 end of the growing DNA chain into a proofreading 35 exonuclease

domain. There, the incorrect nucleotide is removed by the 35 exonuclease activity, whereupon the chain is
moved back into the polymerase domain, where polymerization can continue.
How is fidelity measured?
A variety of polymerase fidelity assays have been described in the literature over the years, perhaps the most
famous being that of Thomas Kunkel (3). The Kunkel method uses portions of the lacZgene in M13
bacteriophage to correlate host bacterial colony color changes with errors in DNA synthesis. Wayne Barnes built
upon this assay and utilized PCR to copy the entire lacZ gene and portions of two drug resistance genes with
subsequent ligation, cloning, transformation and blue/white colony color determination (4). In both assays, errors
incorporated in the lacZ gene cause a disruption in -galactosidase activity leading to a white colony phenotype.
With these lacZ-based experimental approaches, the percentage of white colonies must be converted to the
number of errors per base incorporated. As a more direct read-out of fidelity, Sanger sequencing of individual
cloned PCR products can also score DNA polymerase fidelity and offers the advantage that all mutations will be
detected. Using this method, the entire mutational spectrum of a polymerase can be determined and there is no
need to correct for nonphenotypic changes.
A modification of the lacZ Barnes assay is commonly used at NEB for determination of DNA polymerase fidelity,
as the 1,000 amino acid open reading frame affords a reasonable sequence window for the scoring of DNA
polymerase errors (Figure 2). In this study, results from the lacZ assay were compared to Sanger sequencing to
assess the fidelity of Q5, a new NEB DNA polymerase.