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Practical Manual
   
7. POLYMERASE CHAIN REACTION
The Polymerase Chain Reaction (PCR) is a technique for the in
vitro amplification of specific DNA sequences by the simultaneous primer
extension of complementary strands of DNA. The PCR method was devised and
named by Kary Mullis and colleagues at the Cetus Corporation, although the
principle had been described in detail by Khorana and colleagues over a
decade earlier. The use of PCR was limited until heat stable DNA polymerase
became widely available. The PCR is a major development in the analysis of
DNA and RNA because it has both simplified existing technology and enabled
the rapid development of new techniques, which would not otherwise have been
possible.
DNA polymerases carry out the synthesis of a complementary strand
of DNA in the 5’
to 3’ direction using a single stranded template,
but starting from a double stranded region. This is the primer extension
reaction and is the basis for a variety of labeling and sequencing
techniques.
The Polymerase Chain Reaction (PCR) uses the same principle, but
employs two primers, each complementary to opposite strands of the region of
DNA, which have been denatured by heating. The primers are arranged so that
each primer extension reaction directs the synthesis of a strand of DNA
towards the other. Thus primer
‘a’
directs the synthesis of a strand of DNA which can then be primed by primer
‘b’ and vice versa.
This results in the de novo synthesis of the region of DNA flanked by the
two primers.
The requirements of the reaction are simple:
1.
dNTPs: 200-250
μM
of each dNTP
2.
A pair of synthetic oligonucleotide primers – 0.1 – 0.5
μM
3.
A thermostable DNA polymerase: 0.5 – 2.5U/25-50
μl
reaction
4.
Divalent cations: 1.5 mM Mg2+
5.
Monovalent cations: 50 mM KCl
6.
Buffer: 10 mM Tris Cl – pH 8.3-8.8
7.
Template: DNA
All the above components are mixed in a microfuge tube and the reaction is
carried out in a thermal cycler, which is an automated instrument that takes
the reaction through a series of different temperatures for ranging amount
of time.
Each PCR cycle theoretically doubles the amount of targeted template
sequence in the reaction. Ten cycles theoretically multiply the amplicon by
a factor of about one thousand, 20 cycles, by a factor more than a million
in a matter of hours. In principle and virtually in practice, 2h copies the
duplex segment bordered by primer are produced, where
‘n’
is the no. of cycles. Each cycle of PCR amplification consists of a no. of
steps which produce two oligo nucleotide primed single stranded DNA
template. These steps should be optimized for each template and primer pair
combination.
Step 1 Denaturation
Step 2 Annealing
Step 3 Extension
The next cycle begins with a return to denaturation step and the
cycle continues for a given no.of cycles.
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