Practice question

DpnI is methylation-dependent restriction enzyme recognizing tetranucleotide sequence 5'-GATC-3' only when adenine is methylated at N6 position by bacterial Dam methylase. Plasmid DNA isolated from standard laboratory Escherichia coli strains such as DH5alpha or XL1-Blue is fully methylated at GATC sites, rendering it sensitive to DpnI cleavage. In site-directed mutagenesis, newly synthesized strands generated by proofreading polymerase in vitro lack methylation and resist digestion. DpnI treatment selectively destroys parental template, dramatically reducing wild-type background colonies and enriching desired mutants, with no activity on RNA.