Rapid site-specific mutagenesis in plasmids

Abstract

A quick and simple method for introducing site-specific mutations into plasmids is described. The procedure involves restriction-enzyme digestion of the plasmid to give a linear fragment. A second preparation of the same plasmid is digested with other restriction enzymes to remove the targeted mutational region to give a gapped fragment. The linear fragment and the gapped fragment are mixed, then denatured and annealed in the presence of a short, synthetic oligodeoxynucleotide corresponding to the targeted region and containing the desired mutation. The mix is then transformed directly into cells where host enzymes fill single-stranded gaps to make a complete double-stranded, mutant plasmid.