GATE BT 2019 • Q37
MCQ • 2 Marks
Section 3: Genetics, Cellular and Molecular Biology
Recombinant DNA technology
Subtopic: Fundamentals of Recombinant DNA technology
Question Statement:
For site-directed mutagenesis, which one of the following restriction enzymes is used to
digest methylated DNA?
Options:
-
(A) KpnI
-
(B) DpnI
-
(C) XhoI
-
(D) Mul
Step-by-Step Solution & Explanation
### **Official Solution & Concept Analysis (GATE BT 2019 Q.37)**
**1. Governing Concept**:
- **Topic**: Molecular Biology & Site-Directed Mutagenesis
- In the QuikChange site-directed mutagenesis protocol:
- Plasmid DNA isolated from *E. coli* host is methylated at adenine residues within $5'\text{-GATC-3'}$ sequences by **Dam methylase**.
- PCR amplification using mutagenic primers synthesizes nascent unmethylated mutant strands in vitro.
- To selectively eliminate the wild-type parental template plasmid, the reaction mixture is digested with the restriction endonuclease **DpnI**.
- **DpnI specifically cleaves methylated and hemimethylated $5'\text{-G}^{m}\text{ATC-3'}$ DNA**, leaving the synthesized unmethylated mutant plasmid intact for transformation.
**2. Option Analysis**:
- **Option (A) is INCORRECT**: Cleaves unmethylated DNA, destroying the mutant.
- **Option (B) [DpnI] is CORRECT**: Specifically digests methylated parental plasmid DNA templates while preserving in vitro synthesized mutant strands.
- **Option (C) is INCORRECT**: Standard restriction enzyme non-specific for DAM methylation.
- **Option (D) is INCORRECT**: Mismatched enzyme.
**3. Final Answer**:
- **Correct Choice**: **B**
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