Primer-directed mutagenesis of linearized plasmids☆

Abstract Two novel mutagenesis techniques to specifically alter the sequence of plasmid DNA have been developed. In contrast to other primer-directed mutagenesis methods which require a single-stranded, closed-circular template, a linearized single strand was used as the mutagenesis template. The template is prepared by restriction enzyme digestion of covalently-closed-circular plasmid DNA. These methods are simple, require small amounts of plasmid DNA, and can result in a relatively high frequency of mutagenesis.