For site-directed mutagenesis, which one of the following restriction enzymes is used to digest methylated DNA?
- (A)
KpnI
- (B)
DpnI
- (C)
XhoI
- (D)
MluI
GATE 2019 BT Q.27: solution and answer
Answer and solution
ANSWER
(B) DpnI: it cuts GATC only when the adenine is methylated, so it destroys the methylated parental template left after mutagenesis PCR.
Official answer: BSource: Official answer key, GATE 2019 BT (IIT Madras)
In PCR-based site-directed mutagenesis, the template plasmid is isolated from a dam⁺ strain of E. coli, so the adenine in each of its GATC sites is methylated by Dam methylase.
The mutant strands are made in vitro by PCR, so they carry no Dam methylation.
DpnI cuts GATC only when the adenine is methylated (methylated or hemimethylated DNA). Treating the reaction with DpnI therefore digests the parental template and leaves the unmethylated, mutated DNA intact, which enriches the mutant plasmid.
KpnI, XhoI and MluI are ordinary sequence-specific restriction enzymes; none of them cuts only methylated DNA. The answer is (B) DpnI.
KEY IDEA
In PCR-based site-directed mutagenesis (e.g. QuikChange), the parental plasmid template (isolated from a dam+ E. coli strain) is Dam-methylated at GATC sites, while the newly synthesized mutant strand made in vitro is unmethylated. DpnI is methylation-dependent and cleaves only methylated/hemimethylated GATC, so treating the PCR product with DpnI selectively destroys the parental template and enriches for the mutant plasmid.
WATCH OUT FOR
Confusing DpnI (cuts only methylated DNA) with the many restriction enzymes that are blocked by methylation (cut only unmethylated DNA), or picking a sequence-specific enzyme (KpnI, XhoI, MluI) that has no relationship to methylation status at all.
From the GATE 2019 BT paper. Every question of this paper · Download the full paper · All 3 Editing & interactions questions, with notes