Question

Explain in detail each of the steps of PCR involved in the amplification of a specific region of DNA

Answer 1

PCR-it is an in vitro DNA amplification process in which
millions of copies of a particular sequence of DNA can be produced within a short period of time.

There are mainly 3 steps in PCR,

1)Separation /Denaturation: DNA strands are separated/ melted by heating at 95°C for 15 seconds to 2 minutes.
2)Priming /Annealing:here the primers are annealed by cooling to 50°C for 0.5 to 2 minutes.here the primers hybridise with their complementary single stranded DNA produced in the step 1 of PCR.
3)Polymerization:here new DNA strands are synthesized by Taq polymerase(enzyme is isolated from bacteria Thermus acquaticus which is found in hot springs) so the enzyme is not denatured at high temperature. The polymerization is taking place at 72°C for 30 seconds in presence of deoxyribonucleotide triphosphates(dNTPs). In this manner both strands of DNA are duplicated.
* The steps of 1,2 and 3 are repeated. In each cycle, the DNA strands are doubled. So approximately 20 cycles provide for 1 million times amplifications.(repetition of cycles occurs in Tempcycler instrument).
* After the amplification of desired DNA sequences,the presence of desired DNA can be identified by DNA hybridization technique or Southern blot analysis with a suitable probe.