Question

During the process of gene amplification using PCR, if a 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. Ligation
• D. Denaturation

Answer 1

The correct option is D

Denaturation

The correct option is D.

An explanation for the correct option:

1. In order for the primers to bind to the target area and begin an extension, the double-stranded template DNA must first be separated into single strands during the initial denaturation stage of PCR.
2. Commonly, the first denaturation phase is carried out at 94 to 98 °C for one to three minutes.
3. This step's duration and temperature can change based on the type of template DNA used and the buffer's salt content.

An explanation for the incorrect options:

Option A:

1. The primers must anneal for 30sec to 1 minute at 45 to 60 °C in order to bind to the complementary sequence on each of the DNA single strands.
2. The target of interest is bracketed by the primers, and the section of sequence that lies in between them is known as the amplicon.

Option B:

1. The extension step is the third stage in a PCR cycle.
2. The PCR stage known as the extension step, also known as the elongation step, is where Taq polymerase adds nucleotides to the annealed primer.
3. PCR cycling is the process of repeatedly performing the PCR denaturation, annealing, and extension stages.

Option C:

1. Ligation mixtures can be employed directly as templates, and traditional gel electrophoresis can be utilized to examine the outcomes.
2. The recombinant molecules produced during the ligation and the accompanying transformants are represented by the PCR products.
3. An internal primer can also be used to establish the orientation of inserts.

Final answer: During the process of gene amplification using PCR, if a very high temperature is not maintained in the beginning, then denaturation step of PCR will be affected first