Recombinant Circle Polymerase Chain Reaction for Site-Directed Mutagenesis

Abstract

Site-directed mutagenesis permits modification of the functional characteristics of specific proteins and the characterization of regulatory DNA elements. Since the amplifying primers used in the polymerase chain reaction (PCR) are incorporated into the product, PCR can be used to modify the ends of DNA segments (1,2). Site-directed mutagenesis is accomplished by introducing a mismatch into one of the oligonucleotides used to prime the PCR amplification. This oligonucleotide, with its mutant sequence, is incorporated into the PCR product. Sequences can also be attached to the ends of a DNA segment by using primers whose 5′ ends do not anneal to the original template.