Technical Abstract: We have developed a new, easier-to-use variation of PCR-based site- directed mutagenesis. This highly accurate approach takes advantage of the property of Class IIS restriction enzymes, whose cleavage sites are often separated from the recognition sites. When a recognition site of a Class IIS restriction enzyme is included at the 5' end of a mutagenic PCR primer, ,the recognition sequence is cleaved off, leaving a cohesive end. The othe primer can be designed so that 1) it creates a compatible end when digested with the same enzyme, and 2) it recreates the coding sequence adjacent to the altered bases. This strategy offers a more reliable and easier mode of site-directed mutagenesis than conventional protocols, which involve blunt- end ligation or circular amplification of a plasmid.