SITE-DIRECTED MUTAGENESIS OF DOUBLE-STRANDED DNA BY THE POLYMERASE CHAIN-REACTION

We have developed a facile procedure for rapid PCR-based site-directed mutagenesis of double-stranded DNA. Increasing the initial template c oncentration and decreasing the PCR cycles to 5-10 allows us to reduce the rate of undesired second-site mutations and dramatically increase the time savings. Following PCR, DpnI treatment is used to select aga inst parental DNA molecules. The DpnI (target sequence 5'-Gm(6)ATC) is specific for methylated and hemimethylated DNA and is used to digest parental DNA and select for mutation-containing amplified DNA. DNA iso lated from almost all common Escherichia coli strains is Dam methylate d and therefore susceptible to DpnI digestion. Pfu DNA polymerase is u sed, prior to intramolecular ligation of the linear template, to remov e any bases extended onto the 3' ends of the PCR product by Taq DNA po lymerase. The recircularized vector DNA incorporating the desired muta tions is transformed into E. coli. This method can be used independent ly of any host strain and vector.