Q.37 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

DpnI is the restriction enzyme used to digest methylated DNA in site-directed mutagenesis. This selects for newly synthesized, unmethylated mutant plasmids over the parental template. The correct answer is (B) DpnI.​

Option Analysis

KpnI (A): KpnI recognizes GGTACC and cuts unmethylated or dam-methylated DNA without specificity for methylation status. It serves general cloning but not template removal in mutagenesis.​

DpnI (B): DpnI recognizes GATC and cleaves only fully N6-methyladenine dam-methylated DNA from E. coli. In site-directed mutagenesis, PCR-amplified mutants lack methylation, so DpnI digests parental templates selectively.​

XhoI (C): XhoI cuts CTCGAG regardless of methylation and faces inhibition from overlapping dam sites, not activation by them. It lacks utility for methylated template digestion.​

MluI (D): MluI recognizes ACGCGT and remains unaffected by dam methylation, functioning in standard vector manipulation without selective digestion.​

Site-directed mutagenesis relies on DpnI to digest methylated DNA templates, enabling precise genetic modifications in biotechnology research. This technique introduces targeted changes via PCR, followed by DpnI treatment to eliminate parental plasmids from dam+ E. coli.​

Mechanism in Mutagenesis

PCR uses mutagenic primers on methylated template, yielding unmethylated products. DpnI then cleaves GATC sites only in methylated strands, enriching mutants up to 100% efficiency. Protocols incubate at 37°C overnight post-PCR.​

Why DpnI Over Others?

DpnI’s unique dependence on N6-methyladenine ensures clean selection, critical for protein engineering and functional studies. Researchers in molecular biology favor it for high-fidelity results.​