A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction

Abstract

A specific mutagenic change in the cDNA of human protein S was introduced by a modification of the polymerase chain reaction that permits the introduction of a mutation at any position in a double-stranded DNA molecule. The method employed four synthetic oligonucleotide primers. One oligonucleotide contained a single-base mismatch to direct the mutagenesis; the other three oligonucleotides were designed to allow selective amplification of the mutated sequence with Thermus aquaticus polymerase. The mutagenized cDNA was cloned into a plasmid vector and transformed into Escherichia coli RR1 cells for characterization. The desired cytosine to guanine change in the target cDNA was confirmed by the predicted appearance of an AluI restriction site and by dideoxynucleotide sequencing. No other sequence changes were detected within the amplified region. This method of site-specific mutagenesis can be applied to any linear double-stranded DNA large enough for primer annealing and obviates specialized cloning vectors, DNA constructs, and selection techniques. It has the advantage over a recently published PCR technique ( R. Higuchi, B. Krummel, and R. Saki (1988) Nucleic Acids Res. 16, 7351–7367) in requiring no diafiltration to remove primers between steps and in requiring only a single mutagenic oligonucleotide to be synthesized for each mutant construct made after the initial one.