Explain different steps involved in PCR technique.

Answer: The polymerase chain reaction (PCR), developed by Kary Mullis, amplifies a specific DNA segment in vitro. The reaction mixture contains template DNA, two primers, the four dNTPs and thermostable Taq DNA polymerase. Each cycle has three steps:

1. Denaturation: the mixture is heated to about 94–96 °C; hydrogen bonds break and the double-stranded DNA separates into two single strands.
2. Annealing (primer binding): the mixture is cooled to about 50–60 °C (around 55 °C); the two short oligonucleotide primers bind to the complementary 3′ ends of the single strands.
3. Extension (polymerisation): at about 72 °C, Taq polymerase adds nucleotides to the 3′ end of each primer, synthesising new complementary strands.

The cycle is repeated 25–30 times; the DNA doubles every cycle, so about 2ⁿ copies (over a billion) are obtained. PCR is used in DNA fingerprinting, diagnosis of diseases and gene cloning.