Site-directed mutagenesis using thermostable enzymes.

The two-primer method for site-directed mutagenesis facilitates the mutation of targets in double-stranded DNA. We have encountered difficulties using the original method for the mutagenesis of DNA cloned into pBluescript vectors, which is possibly due to the presence of regions of secondary structure that cannot be efficiently copied by the enzymes used. We report a modification of the two-primer method using the thermostable VentR DNA polymerase and Thermus aquaticus ligase, allowing an increase in reaction temperatures. The modified method is functional with the pBluescript family of vectors.