A RAPID METHOD FOR SITE-SPECIFIC MUTAGENESIS USING LARGER PLASMIDS ASTEMPLATES

To facilitate the introduction of specific point mutations in plasmids that are too large to be amplified efficiently by a single PCR, we ha ve developed a method for site-directed mutagenesis by generating part ial plasmid fragments, which introduces changes as simply as conventio nal techniques. Plasmids containing a fragment of the human immunodefi ciency virus type-1 (HIV-1) envelope gene were subjected to PCR with f our pairs of PCR primers for each desired point mutation. One primer i n each of two of these four pairs contained the desired mutation. The four pairs of primers were designed so that four overlapping fragments were amplified from the plasmid template, two of which contained the mutation. These fragments were then reannealed and electroporated dire ctly into Escherichia coli. The desired mutation was typically found i n 66% to 83% of the resulting colonies. The technique is almost as sim ple as previous techniques, shows similar efficiency and is applicable to plasmids that would normally be too large for efficient site-speci fc mutagenesis. The entire procedure, from PCR amplification to transf ection into E. coli, can be completed in one day.