Generate a Mutant Gene Using External and Internal Primers
How can we generate a mutant gene using external and internal primers?
What are the steps involved in the process?
Answer:
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
Explanation:
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.