Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed...

A new method is described for rapid site-directed mutagenesis of plasmid DNA. The new method, termed enzymatic inverse polymerase chain reaction (EIPCR), uses inverse PCR to amplify the entire plasmid. The key step to EIPCR is the incorporation of identical class 2s restriction sites in both primers. Class 2s restriction enzymes have a recognition site that is located 5' of the cut site (e.g., BsaI: GGTCTCN'NNNN,). Thus, after completing PCR, the ends of the full-length linearized plasmid are digested with the class 2s enzyme incorporated into the primers. The enzyme cuts off its entire recognition site and leaves the plasmid with compatible overhangs on both ends. Thus, in the ligation the only part that becomes part of the plasmid is the NNNN overhang, which can be made to be the native sequence. We have used the method for many plasmids and several class 2s enzymes. As an example, we report here the use of EIPCR for an insertion into pUC19 containing an inactive lacZ alpha-peptide, causing a frameshift that restores lacZ alpha-activity. Of 300 colonies evaluated, greater than 95% had the expected blue phenotype. The BsaI overhangs were correctly combined in all of the 35 blue colonies analyzed by restriction digestion and in all four clones that were sequenced. EIPCR is compared with four related PCR-based mutagenesis techniques. The major advantage of EIPCR over the other methods is the combination of greater than 95% correctly mutated clones with the need for only two PCR primers.