A Simple Method for Site-Specific Mutagenesis that Leaves the Rest of the Template Unaltered

Site-directed mutagenesis is an important component of modern molecular biology. The use of this technique became necessary both in our studies of promoter function and in the analysis of protein structure-function relationships. Although several polymerase chain reaction (PCR)-based methods of site-specific mutagenesis have been described (1 –4 ), we have developed a simple procedure that can introduce either a single or a small cluster of mutations into any DNA segment in the range 0.3–3 kb. Importantly, using this technique, only the designed changes are incorporated into the target sequence, all other positions remain unaltered.