Question
Arrange the following steps of site-directed mutagenesis in correct order :
A. Transformation of ds DNA into cells
B. Denaturation and annealing of oligonucleotide primers with mutation
C. Repair of nicks in mutated plasmid inside cells
D. Extension and incorporation of primers by using DNA polymerase
E. Digestion of unmutated parental DNA template with methylation specific nuclease and annealing of newly synthesized strands
Choose the correct answer from the options given below :
This question was previously asked in
CUET PG 2026 Agri-Business Management Question Paper (25-Mar-2026) (Shift 2)
The correct answer is
B, D, E, A, C
Site-Directed Mutagenesis Steps Explained
To correctly order the steps of site-directed mutagenesis, we follow the logical progression of the molecular process:
- Denaturation and annealing of oligonucleotide primers with mutation (B): This is the crucial first step where the synthetic DNA primers, designed to contain the desired mutation, are hybridized to the template DNA.
- Extension and incorporation of primers by using DNA polymerase (D): A DNA polymerase enzyme extends the primers, synthesizing a new DNA strand complementary to the template. This incorporates the mutation into the newly synthesized strand, creating a heteroduplex molecule.
- Digestion of unmutated parental DNA template with methylation specific nuclease and annealing of newly synthesized strands (E): In many protocols, the original, non-mutated template DNA (often methylated) is selectively degraded using specific nucleases (like DpnI) to eliminate it and enrich the population of the desired mutated DNA.
- Transformation of ds DNA into cells (A): The processed, mutated DNA is then introduced into competent host cells (e.g., bacteria) via transformation.
- Repair of nicks in mutated plasmid inside cells (C): Once inside the host cell, the cellular DNA repair machinery acts on the plasmid DNA, sealing any nicks and ensuring the stable replication of the mutated plasmid.
Therefore, the correct sequence representing the process of site-directed mutagenesis is B, D, E, A, C.
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