Question

Explain different steps involved in PCR technique.

Hint:

Remember the PCR cycle as a simple 3-step recipe: \textbf{1. Heat} to separate (Denature). \textbf{2. Cool} to prime (Anneal). \textbf{3. Warm} to copy (Extend).

Answer 1

PCR, or Polymerase Chain Reaction, is a laboratory technique used to make millions of copies of a specific segment of DNA. Each cycle of PCR involves three main steps:

Denaturation:

The reaction mixture, containing the target DNA, primers, nucleotides, and DNA polymerase, is heated to a high temperature, typically 94-96°C.
This high heat breaks the hydrogen bonds holding the two strands of the DNA double helix together, causing the DNA to separate into two single strands. Each single strand can now serve as a template for the synthesis of a new strand.

Annealing:

The temperature of the reaction is lowered to about 50-65°C. The exact temperature depends on the sequence of the primers.
At this temperature, short, single-stranded DNA sequences called primers bind (anneal) to their complementary sequences on the single-stranded template DNA. Primers are necessary as they provide a starting point for the DNA polymerase.

Extension (or Elongation):

The temperature is raised again to around 72°C, which is the optimal temperature for the heat-stable DNA polymerase enzyme (like Taq polymerase) to function.
The DNA polymerase attaches to the primers and moves along the template strand, adding complementary nucleotides (A, T, C, G) to synthesize a new DNA strand. This results in two new double-stranded DNA molecules.

This three-step cycle is repeated 25-35 times, leading to an exponential amplification of the target DNA sequence.