POLYMERA

SE
CHAIN
REACTION

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POLYMERASE CHAIN REACTION

Polymerase chain reaction (PCR) is a method that allows logarithmic amplification of
short DNA sequences within a longer double stranded DNA molecule.
PCR SET-UP:
1. Template.
2. Primer - Forward and reverse.
3. dNTP's.
4. Taq polymerase.
5. Taq polymerase buffer.
6. Nuclease free water.
7. Additives (optional).
TEMPLATE:
DNA isolated from any organism may serve as a template.
PRIMER:
PCR entails the use of a pair of primers, each about 20 nucleotides in length,
that are complementary to a defined sequence on each of the two strands of the
DNA.
dNTP's (Deoxyribonucleotide phosphates):
It is a mixture of nucleotides like dATP, dGTP, dTTP, dCTP.
Taq POLYMERASE:
A thermo-stable DNA polymerase
Isolated from Thermus aquaticus, a bacterium that grows in hot pools.
It is not necessary to add new polymerase in every round of amplification.
ADDITIVES:
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 Certain additives are required to improve the quality of PCR process.

PROCEDURE:
The PCR process usually consists of a series of twenty to thirty-five cycles. Each
cycle consists of three steps:
Denaturation:
The double-stranded DNA has to be heated to 94-96C (or 98C if
extremely thermostable polymerases are used) in order to separate the
strands.
Annealing:
After separating the DNA strands, the temperature is lowered so the
primers can attach themselves to the single DNA strands.
The temperature of this stage is around 45-65C.
Extension:
The elongation temperature depends on the DNA polymerase.
The DNA polymerase has to copy the DNA strands. It starts at the
annealed primer and works its way along the DNA strand.
Taq polymerase elongates optimally at a temperature of 72C.
A final elongation step is frequently used after the last cycle to ensure that
any remaining single stranded DNA is completely copied.
TYPES OF PCR:
1. Standard PCR/ Quantitative PCR.
2. Multiplex PCR.
3. Nested PCR.
4. RT-PCR (Reverse Transcriptase PCR).

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5. Hot start PCR.

ERRORS IN PCR AND TROUBLE SHOOTING:

Polymerase chain reaction is not perfect, some common errors and problems may
occur. They are listed below:
POLYMERASE ERRORS:
Taq polymerase lacks a 3' to 5' exonuclease activity (Proof reading activity).
The rate of error is approximately 1 in 10,000 bases, which, can alter large
proportions of the final product.
Solution:
Vent, which is extracted from Thermococcus litoralis; Pfu DNA polymerase, which is
extracted from Pyrococcus furiosus and Pwo DNA polymerase, which is extracted
from Pyrococcus woesii.
SIZE LIMITATIONS:
PCR works readily with DNA of lengths two to three thousand basepairs, above this
length the eficiency of the reaction reduces.
The polymerase activity is markedly reduced.
Solution:
It is possible to amplify larger pieces of up to 50,000 base pairs with a slower heating
cycle and special polymerases.
NON SPECIFIC PRIMING:
The non specific binding of primers is always a possibility due to sequence
duplications, non-specific binding and partial primer binding, leaving the 5' end
unattached.
Solution:
Non-specific priming can be prevented during the reaction by the preparation of "hotstart" polymerase enzymes or using nested primers.

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DESCRIPTION:
Quantitative PCR:
Realtime or quantitative PCR (QPCR) allows quantification of starting amounts of
DNA, cDNA, or RNA templates.
QPCR is based on the detection of a fluorescent reporter molecule that increases as
PCR product accumulates with each cycle of amplification.
Fluorescent reporter molecules include dyes that bind double-stranded DNA (i.e.
SYBR Green I) or sequencespecific probes (i.e.TaqMan Probes).
Multiplex PCR:
Multiplex PCR is a variant of PCR which enabling simultaneous amplification of
many targets of interest in one reaction by using more than one pair of primers.
This method has been applied in many areas of DNA testing, including analyses of
deletions, mutations, and polymorphisms.
Nested PCR:
Nested PCR means that two pairs of PCR primers were used for a single locus.
The first pair amplified the locus as seen in any PCR experiment.
The second pair of primers (nested primers) binds within the first PCR product and
produces a second PCR product that will be shorter than the first one.
Any mistake during the first cycle could be rectified by using a second set of internal
primer. This increases the specificity of the product amplified.
RT-PCR (Reverse Transcriptase PCR):
RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive
technique for mRNA detection and quantitation currently available.
RT-PCR can be used to quantify mRNA levels from much smaller samples.
Hot start PCR:
Hot Start PCR techniques focus on the inhibition of polymerase activity during
reaction preparation.
By limiting polymerase activity prior to PCR cycling, non-specific amplification is
reduced and target yield is increased.

APPLICATIONS OF PCR:
PCR is generally used in the following areas of research:
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1. Identification and discrimination of bacterial strains.

2. Detection of hereditary diseases.
3. Identification of genetic fingerprints.
4. Diagnosis of infectious disease.
5. Cloning of genes.
6. Paternity testing.
7. Forensic application.
DIAGRAMATIC REPRESENTATION OF POYMERASE CHAIN REACTION

A- DENATURATION
EXTENSION

B- ANNEALING

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C-