Generate a Mutant Gene Using External and Internal Primers

10 Sep 2023

How can we generate a mutant gene using external and internal primers?

What are the steps involved in the process?

The steps to generate a mutant gene using two external and two internal primers are:

1. Primer design

2. PCR reaction setup

3. Denaturation

4. Annealing

5. Extension

6. PCR cycling

7. Verification

Primer Design: Design two external primers that flank the gene of interest and two internal primers that introduce specific mutations.

PCR Reaction Setup: Prepare a PCR reaction mix containing DNA template, external primers, internal primers, nucleotides, and DNA polymerase.

Denaturation: Heat the reaction mix to a high temperature to denature the DNA strands.

Annealing: Cool the reaction mix to a lower temperature to allow the primers to bind to the DNA template.

Extension: Raise the temperature to allow DNA polymerase to extend the primers and synthesize new DNA strands.

PCR Cycling: Repeat the denaturation, annealing, and extension steps for multiple cycles to amplify the DNA.

Verification: Analyze the PCR product using gel electrophoresis or DNA sequencing to confirm the presence of the mutant gene.