PCR Site-Directed Mutagenesis Using Pyrococcus sp GB-D Polymerase Coupled to a Rapid Screening Procedure: Application to a -Glucanase Gene

PCR methodology is one of the fastest available procedures for site-directed mutagenesis (1 ,2 ). However, it has been criticized for a lack of reliability because of unwanted mismatches produced during the PCR reaction (3 ,4 ). In the present protocol, we describe an improvement on the efficiency of site-directed mutagenesis by PCR using the Pyrococcus species GB-D polymerase instead of the commonly used Thermus aquatiqus (Taq) polymerase. Taq polymerase lacks a 3′→5′ proofreading exonuclease activity that is not crucial for several PCR applications, but is advisable for site-directed mutagenesis experiments. Some thermophilic DNA polymerases have this activity, among them the Thermococcus litoralis and the Pyrococcus species GB-D enzymes. A 10-fold higher efficiency has been reported for these enzymes over that observed for Taq polymerase (5 ). PCR site-directed mutagenesis is specially suitable for protein engineers when it is coupled to a screening procedure directly performed on the transformant plates. In such cases the procedure is rapid (3 d from mutagenic primers to selection of clones) and efficient (98–100% of successful mutagenesis).