Question

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?

• A. Annealing
• B. Extension
• C. Denaturation
• D. Ligation

Answer 1

The Correct Option is C

Polymerase Chain Reaction (PCR) is an essential molecular biology technique used to amplify DNA sequences. The PCR process involves three key steps: denaturation, annealing, and extension. Let us understand each step's role: 

1. Denaturation: This step involves heating the reaction mixture to a high temperature (usually around \(94^\circ\text{C}\) to \(98^\circ\text{C}\)) to separate the double-stranded DNA into single strands. This is critical as it allows the primers to access the template DNA for the next steps.
2. Annealing: In this step, the temperature is lowered (typically between \(50^\circ\text{C}\) and \(65^\circ\text{C}\)), allowing the primers to bind or anneal to their complementary sequences on the single-stranded DNA templates.
3. Extension: At this stage, the temperature is adjusted (usually around \(72^\circ\text{C}\)) to enable the DNA polymerase enzyme to add nucleotides to the primed sequences, synthesizing new strands of DNA.

In the context of the given question, if a very high temperature is not maintained at the beginning of the PCR process, the Denaturation step will be affected first. Without adequate heat, the hydrogen bonds between DNA strands won't break effectively, preventing the formation of single-stranded DNA. This failure will obstruct primers' access to the template strand, ultimately inhibiting the entire PCR process.

Now, let's evaluate the options:

• Annealing: Occurs after denaturation; it won't start properly if denaturation isn't complete.
• Extension: Dependent on correct annealing and therefore also on successful denaturation.
• Denaturation: Directly affected by inadequate initial heating. This is the correct answer.
• Ligation: Not a part of the PCR process; it refers to joining DNA strands, which typically occurs outside of PCR, like in cloning.

Thus, the correct answer is Denaturation, as it is directly impacted by not maintaining a high initial temperature in PCR.