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

Polymerase Chain Reaction (PCR) 

Purpose: PCR is a powerful technique used to amplify a specific gene or DNA segment in vitro, producing millions of copies from a small initial sample.

 Steps of PCR:

1. Denaturation:
   The DNA sample is heated to 94–98°C. This breaks the hydrogen bonds between the strands, converting double-stranded DNA into two single strands.
2. Annealing:
   The temperature is reduced to 50–65°C to allow short, single-stranded DNA primers to bind (anneal) to their complementary sequences on the template strands.
3. Extension:
   The temperature is increased to about 72–80°C, optimal for the thermostable enzyme Taq polymerase. This enzyme synthesizes the new DNA strand by adding nucleotides to the primers.
4. Amplification (Cycles):
   The above three steps are repeated for 20–40 cycles, resulting in an exponential increase in the number of DNA copies. After n cycles, ideally: \[ \text{Number of copies} = 2^n \]

✅ Applications:

• Genetic testing and diagnostics
• Forensic science (DNA fingerprinting)
• Infectious disease detection
• Gene cloning and research