PCR site-directed mutagenesis using Pyrococcus sp GB-D polymerase coupled to a rapid screening procedure. Application to a beta-glucanase gene.

Abstract

AbstractPCR 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).KeywordsMutagenic PrimerFlank PrimerRapid Screening MethodMaster PlateGentle AspirationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.