Question

Explain the mechanism of PCR.

Answer 1

1. The PCR technique is employed for in vitro gene cloning or gene multiplication. It uses automatic heat cycles to denaturate and re-denaturate double-stranded DNA.
2. In the Eppendorf tube, the desired DNA segment is mixed with an excess of two primer molecules, four types of DNTPs, and thermostable DNA polymerase. The combination goes through three operations: denaturation, annealing, and primer extension.
3. Denaturation is the process of heating the reaction mixture to 90-98°C in order to separate the two strands of DNA.
4. Annealing is the process of chilling the mixture to 40-60% to allow primers to link with complementary sequences in DNA. The thermostable DNA polymerase extends primers at 70-75°C.
5. It starts with a single strand of DNA as a template and then adds additional nucleotides to the primer.
6. Gel electrophoresis is used to purify the amplified DNA segments.