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 ?

The correct option is Denaturation.

To understand why denaturation is affected first, let us break down the steps involved in the Polymerase Chain Reaction (PCR) process:
PCR is a molecular biology technique used to amplify a specific segment of DNA, generating millions of copies. The process consists of three main thermal steps in each cycle:

1. Denaturation: This is the first step of the PCR cycle. In this step, the double-stranded template DNA is heated to a very high temperature (typically between 94 °C to 98 °C). The high temperature disrupts the hydrogen bonds holding the complementary bases of the two strands together, thereby separating the double-stranded DNA into two single strands.
2. Annealing: In the second step, the temperature is lowered (usually to 50 °C - 65 °C) to allow short DNA sequences called primers to bind (anneal) to their complementary sequences on the single-stranded template DNA.
3. Extension: In the final step, the temperature is raised to an optimal temperature (typically around 72 °C) for the DNA polymerase enzyme (such as Taq polymerase) to extend the primers by adding complementary dNTPs, synthesizing a new DNA strand.

If a very high temperature is not maintained at the beginning of the PCR cycle, the double-stranded DNA will fail to separate because the hydrogen bonds between the strands will not break. Since denaturation is the very first step in the cycle and requires the highest temperature, it will be the first step to be affected. Without successful denaturation, the subsequent steps of annealing and extension cannot occur.