Practice question

In inverse PCR-based site-directed mutagenesis, DpnI endonuclease plays crucial role in selection of newly synthesized mutant plasmids. DpnI specifically recognizes methylated GATC sequences, which occur frequently in parental plasmid DNA propagated in dam+ E. coli strains. Unmethylated PCR product carrying mutation is resistant to DpnI cleavage, whereas methylated parental strands are digested into fragments. This selective removal enriches mutant clones and reduces background wild-type colonies after transformation. DpnI does not cleave mutant DNA, synthesize RNA, or ligate fragments; its function is methylation-dependent restriction for background elimination.