Question

Question of medical biology and genetics: What is PCR (polymerase chain reaction)? How does PCR work? What happens at each of pcr? Explain in details.

Answer 1

PCR stands for Polymerase chain reaction which is an important molecular biological tool discovered by Kary Mullis and his associates. The technique is used for invitro cloning/Amplification of DNA segments for use in recombinant DNA technology or other molecular biological approaches like DNA sequencing, DNA fingerprinting etc. The technique became so popular that it came to be known as People's Choice Reaction.

PCR involves the use of a machine called Thermocyclers and in this thermocycler, all the procedure of DNA amplification is done in a fully automated manner. The reaction mixture for PCR amplification is Sample DNA, dNTPs (dATPs, dGTPs, dCTPs and dTTPs), thermostable DNA polymerase (Taq polymerase), DNA primers and a buffer solution.

The working of PCR involves the following stages:

1. Denaturation

2. Annealing

3. Extension or Elongation

1. Denaturation: In this step, The double stranded DNA is denatured by increasing the temperature of the thermostat to 94^oC or higher. The step breaks the hydrogen bonds between the two strands of DNA (melting) and as a result the two strands of DNA separate and the bases are exposed.

2. Annealing: In this step, the temperature is lowered to to 60- 65^oC, so that the two primers bind to two DNA strands for initiation of DNA replication.

3. Extension or Elongation: In this step, the temperature is raised to 75 - 80^oC so that DNA polymerase synthesizes a new strand of DNA with the help of dNTPs present in the reaction mixture.

In Ist cycle, one DNA molecule produces two DNA molecules, then two to four DNA molecules in the 2nd cycle and 4 to 8 DNA cycle in the 3rd cycle and so on.