Douglas H. Jones

Site-specific mutagenesis and DNA recombination by using PCR to generate recombinant circles in vitro or by recombination of linear PCR products in vivo

This article describes two methods in which the polymerase chain reaction (PCR) is used for site-specific mutagenesis and for DNA recombination without any enzymatic reaction in vitro apart from DNA amplification. The first method generates DNA joints in vitro by using separate PCR amplifications to generate products that when combined, denatured, and reannealed form double-stranded DNA with single-stranded ends. These single-stranded ends are designed to anneal to each other to yield circles, an application termed recombinant circle PCR (RCPCR). RCPCR-generated DNA circles form without restriction enzyme digestion or ligation and can be transfected directly into Escherichia coli . The second method generates DNA joints in vivo by using the polymerase chain reaction to add homologous ends to DNA. Following transfection of the linear PCR product(s) into strains of E. coli used routinely in cloning, recombination of these homologous ends in vivo permits cloning of the mutant or recombinant of interest. The second method, termed recombination PCR (RPCR), diminishes the number of primers necessary to generate a given mutant or recombinant to half that necessary in RCPCR, because it eliminates the need to generate staggered ends in vitro .

Use of polymerase chain reaction for making recombinant constructs.

The capacity to recombine and modify DNA are underpinnings of the recombinant DNA revolution. The polymerase chain reaction (PCR) (1,2) provides a rapid means for the site-directed mutagenesis of DNA and for the recombination of DNA (1-9). Recently, two methods have been introduced that permit site-directed mutagenesis and DNA recombination without any enzymatic reaction in vitro apart from DNA amplification (5-9). The first method is accomplished by using separate PCR amplifications to generate products, such that when these products are combined, denatured, and reannealed, they form doublestranded DNA with single-stranded ends that are designed to anneal to each other to yield circles, an application termed recombinant circle PCR (RCPCR; see Chapter 27 ).