Practice question

Parental plasmids propagated in most Escherichia coli strains are methylated by Dam methyltransferase at N6 position of adenine within sequence GATC. After inverse PCR mutagenesis, mixture contains both methylated wild-type template and unmethylated mutated amplicon. Selective removal of parental background exploits methylation sensitivity. DpnI specifically recognizes GATC only when adenine is methylated, introducing double-strand breaks and fragmenting template into non-functional pieces. Newly amplified mutated DNA lacking methylation resists digestion and remains intact for transformation. This enzymatic selection dramatically improves efficiency, explaining why DpnI treatment is indispensable for site-directed mutagenesis protocols.