A RAPID AND EFFICIENT ONE-TUBE PCR-BASED MUTAGENESIS TECHNIQUE USING PFU DNA-POLYMERASE

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the mega primer PCR mutagenesis approach can be performed in one tube in less t han 4.5 hours, and does not require purification of intermediate produ cts. High fidelity of DNA sequence replication is obtained by employin g Pfu DNA polymerase and limiting the total number of amplification cy cles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction diges t or sequencing techniques.