Practice question

Inverse PCR based site-directed mutagenesis introduces predetermined nucleotide changes using complementary mutagenic primers. Entire circular plasmid is amplified with high-fidelity polymerase using primers carrying desired substitution, generating linear full-length copies containing mutation. Parental plasmid propagated in Dam methylase positive Escherichia coli contains methylated GATC sequences, while PCR product remains unmethylated. Reaction is digested with methylation sensitive DpnI to fragment parental template, leaving mutated amplicons intact. Digested product is transformed into competent cells where it recircularizes. Simplified workflow follows PCR, then DpnI digestion, then transformation.