A SIMPLE AND EFFICIENT METHOD FOR SITE-DIRECTED MUTAGENESIS WITH DOUBLE-STRANDED PLASMID DNA

A general, simple and efficient method for preparing site-specific mut ations in double-stranded plasmid DNA without the need for special pla smids, bacterial strains or reagents is described. Only one synthetic oligonucleotide for each mutation is required, subcloning is unnecessa ry and a high efficiency of mutation (58-97%) was obtained. If two syn thetic oligonucleotide primers are used, two separate mutations can be simultaneously created in a single reaction tube.