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 :

• A. \(B, D, E, A, C\)
• B. \(E, D, B, C, A\)
• C. \(C, B, E, D\)
• D. \(A, C, D, E, B\)

Hint:

Remember the key steps in site-directed mutagenesis: \[ \text{Primer Annealing} \rightarrow \text{DNA Synthesis} \rightarrow \text{Template Digestion} \rightarrow \text{Transformation} \]
• DpnI digests methylated parental DNA.
• Mutagenic primers introduce the desired mutation.

Answer 1

The Correct Option is A

Concept: Site-directed mutagenesis is a molecular biology technique used to introduce specific mutations into a DNA sequence. The process generally involves:
• Use of mutation-containing primers
• DNA synthesis using DNA polymerase
• Removal of parental template DNA
• Transformation into host cells
• Repair and propagation inside cells A common example is PCR-based mutagenesis using methylation-sensitive digestion of the parental DNA.

Step 1: First step — Primer annealing after denaturation. Initially:
• Double-stranded DNA is denatured.
• Mutagenic oligonucleotide primers anneal to the template. Thus, the first step is: \[ \boxed{B} \]

Step 2: Second step — DNA synthesis by polymerase. After primer annealing: \[ \text{DNA polymerase extends the primers} \] forming newly synthesized mutated DNA strands. Thus, the next step is: \[ \boxed{D} \]

Step 3: Third step — Removal of parental template DNA. The original parental DNA is methylated and can be selectively digested using a methylation-specific nuclease such as: \[ \text{DpnI} \] This leaves the newly synthesized mutated DNA intact. Thus, the next step is: \[ \boxed{E} \]

Step 4: Fourth step — Transformation into host cells. The mutated double-stranded DNA is then introduced into bacterial cells. Thus: \[ \boxed{A} \]

Step 5: Final step — Repair of nicks inside cells. Inside the host cell:
• Remaining nicks in plasmid DNA are repaired.
• Mutated plasmid replicates normally. Thus: \[ \boxed{C} \] Therefore, the correct order is: \[ \boxed{B \rightarrow D \rightarrow E \rightarrow A \rightarrow C} \] Hence, the correct answer is: \[ \boxed{(A)\ B, D, E, A, C} \]