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

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