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PCR

Polymerase chain reaction or PCR is a technique developed by American biochemist Kary Mullis to amplify a DNA of interest. In a conventional PCR reaction, the first step is heat-denaturation of the double-stranded DNA at 94 to 96C. This is followed by annealing of the specifically designed primer at the target site and then polymerase catalyzed elongation by the addition of the nucleotides. The denaturation takes about 30 secs, the annealing about 20 to 40 secs and the elongation takes several minutes depending upon the number of cycles set in the PCR machine. PCR machine, DNA template, a polymerase enzyme (usually the heat-resistant Taq polymerase), specifically designed forward and reverse primers, the deoxynucleoside triphosphates or dNTPs mixture which will provide the nucleotides for elongation - they act as the building blocks of the new strand, and the buffer solution to maintain the pH, are the key requirements. The reaction is carried out in 0.1 ml / 0.2 ml PCR tubes or specially designed PCR plates. PCR Kits are also available with everything readymade and quick to use.


In addition to the conventional PCR, a gradient PCR with a range of variation in annealing temperature can also be used. It is Ideal when while developing the protocol for a new DNA sample. Quantitative PCR (qPCR) which uses fluorescent dyes like Sybr Green can be performed to measure the quantity of amplified product in real time.

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