Polymerase chain reaction

1. Polymerase Chain
Reaction
by
K. Ravichandran

2. Definition:
PCR - It is a technique used to amplify a segment of
DNA of interest or produce lots and lots of copies. It is an
enzymatic method and carried out invitro. PCR technique
was developed by Kary Mullis in 1983. It is very simple,
inexpensive technique for characterization, analysis and
synthesis of specific fragments of DNA or RNA from
virtually any living organisms.

3. How does PCR work?
 PCR mimics what happens in cells when DNA is copied (replicated) prior
to cell division, but it is carried out in controlled conditions in a laboratory.
The machine that is used is simply called a PCR machine or a thermocycler.
 Standard ingredients in the mixture are:
 the DNA segment of interest
 specific primers
 heat-resistant DNA polymerase enzyme
 the four different types of DNA nucleotides
 the salts needed to create a suitable environment for the
enzyme to act.

4. What is the PCR process PCR involves a process of heating and
cooling called thermal cycling which is carried out by machine.
There are three main stages:
Denaturing – when the double-stranded template DNA is heated to
separate it into two single strands.
Annealing – when the temperature is lowered to enable the DNA
primers to attach to the template DNA.
Extending – when the temperature is raised and the new strand of
DNA is made by the Taq polymerase enzyme.

5.  Denaturation  The reaction mixture is heated to 95°C for a short time
period (about 15–30 sec) to denature the target DNA into single strands that
can act as templates for DNA synthesis.
 Primer annealing  This annealing temperature is calculated carefully to
ensure that the primers bind only to the desired DNA sequences (usually
around 55oC).
 Extension  The temperature of the mixture is raised to 72°C (usually) and
kept at this temperature for a pre-set period of time to allow DNA
polymerase to elongate each primer by copying the single-stranded
templates. Taq polymerase is the enzyme commonly used for primer
extension, which occurs at 72°C. This enzyme is used because of its ability to
function efficiently at elevated temperatures and to withstand the denaturing
temperature of 94°C through several cycles.

7. Factors affecting PCR
 Primer » PCR reaction needs two primer, a forward and a reverse
primer. Primer are synthesized oligonucleotide usually ranging from
15-30 bases long. Primers are designed such that at 3’end they do not
have more than two bases complementary to each other as this
results in PRIMER-DIMER formation.
•The G+C contents is in the range of 40-60%
•The melting temperature (Tm) of both forward and reverse primer is
usually the same.
•Low concentration of primer results in poor yield while high
concentration may results in non specific amplification. Hence optimal
concentration of primer is needed ie 0.1-1µ

8. ii. Amount of Template DNA
•Optimal amount of template DNA usually in nano gram. Higher
concentration inhibit or results in non specific amplification.
•Taq DNA polymerase:
•Taq DNA polymerase is 94 KD thermostable DNA polymerase isolated
from Thermus aquaticus.
•Optimal temperature for activity of Taq polymerase is 72° but it can tolerate
high temperature and donot affects by denaturating temperature of 94°C.
•Taq DNA polymerase have both 5’-3’ polymerase activity and 5’-3’
exonuclease activity. But it lacks 3’-5’ exonuclease activity (proof reading
activity).

9. Applications:
1.Forensic science: DNA finger printing, paternity testing and
criminal identification
2.Diagnosis: Molecular identification of microorganisms
3.Evolution study: evolutionary biology
4.Fossil study: Paleontology
5.Gene cloning and expression
6.Gene sequencing
7.Vaccine production by recombinant DNA technology
8.Drug discovery
9.Mutation study
10.Human genome project