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Question
What is PCR? Explain different steps involved in it.
Solution
Definition: Polymerase Chain Reaction (PCR) is the process of in vitro amplification of gene of interest using a PCR machine.
Mechanism of PCR: At the start of PCR, all the requirements are mixed together in ‘eppendorf tube’ and the following operations are performed sequentially:
Step i: Denaturation
The reaction mixture is heated to a temperature (90–98o C) to separate two strands of desired DNA. This is called denaturation.
Step ii: Annealing
The mixture is allowed to cool (40–60o C) that permits pairing of the primer to the complementary sequences in DNA. This step is called annealing.
Step iii: Primer extension / Polymerization
The temperature (70–75o C) allows thermostable Taq DNA polymerase to use single-stranded DNA as template and adds nucleotides. This is called primer extension. It takes around two minutes duration.
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