During the process of gene amplification using PCR, if very high temperature is not maintained in the beginning, then which of the following steps of PCR will be affected first?

Polymerase Chain Reaction (PCR) consists of three cyclic steps that are repeated many times:

1. Denaturation – heating the reaction mixture to a very high temperature (usually around 94–98 °C) to melt the double‑stranded DNA into single strands.

2. Annealing – cooling the mixture (typically 50–65 °C) so that short primers can bind (anneal) to their complementary sequences on the single‑stranded DNA.

3. Extension – raising the temperature to the optimal working temperature of the DNA polymerase (around 72 °C) so that the enzyme extends the primers, synthesizing new DNA strands.

If the very high temperature required for the first step is not maintained, the double‑stranded DNA will not fully melt. This incomplete melting prevents the creation of single‑stranded templates, which are essential for primers to bind in the subsequent annealing step. Consequently, the very first step that is affected when the high temperature is lacking is the Denaturation step.