Question

Explain the steps of amplification of gene of interest using PCR technique.

Hint:

In PCR, each cycle doubles the amount of DNA, leading to exponential amplification. The key to success in PCR is using a heat-stable polymerase enzyme like Taq polymerase.

Answer 1

The Polymerase Chain Reaction (PCR) technique is used to amplify a specific gene of interest. The steps are as follows:
1. Denaturation: The DNA sample is heated to about 94-98°C to denature the DNA, causing the double-stranded DNA to separate into two single strands.
2. Annealing: The temperature is lowered to about 50-65°C to allow short DNA primers to bind (anneal) to the complementary sequences on the single-stranded DNA.
3. Extension: The temperature is raised to around 75-80°C, which is the optimal temperature for the enzyme Taq polymerase to extend the primers and synthesize the new DNA strand.
4. Amplification: These steps are repeated for 20-40 cycles, leading to exponential amplification of the target gene.