A HIGHLY EFFICIENT PROCEDURE FOR SITE-SPECIFIC MUTAGENESIS OF FULL-LENGTH PLASMIDS USING VENT DNA-POLYMERASE

Careful titration of Vent polymerase activity allows efficient amplifi cation of full-length plasmids (12 kb). The high processivity and fide lity of this enzyme made oligonucleotide-directed site-specific mutage nesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant pl asmids were obtained consistently with > 90% efficiency from a single round of PCR. This procedure also made site-specific deletion, inserti on, and several bases mutagenesis facile and efficient.