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 ).