Practice question

In inverse PCR mutagenesis, reaction mixture contains parental methylated plasmid propagated in Dam positive bacteria and newly synthesized unmethylated mutated copies produced by high-fidelity polymerase in vitro. To enrich mutants, mixture is incubated with DpnI restriction enzyme which recognizes methylated GATC sites present only in parental template, introducing double-strand breaks and fragmenting it. This selective digestion leaves unmethylated mutated amplicons intact, drastically lowering wild-type background upon transformation. Treatment therefore digests template DNA while retaining mutated copies, rather than cutting all DNA or degrading RNA.