In Vitro Recombination and Mutagenesis by Overlap Extension PCR

The polymerase chain reaction (PCR) (1 ,2 ) is now a fundamental tool of molecular biology. Although PCR provides the basis for a variety of sensitive analytical techniques, it can also be used in a synthetic capacity to generate large quantities of specific DNA fragments. The alteration of amplified DNA sequences is also possible, since synthetic oligonucleotide primers become incorporated into the final PCR product. Although the 3′ ends of these primers must match the target DNA sequence, the 5′ ends may contain modifications. Sequence modifications in the primers will therefore be present in the ends of the amplified DNA fragment, offering a straightforward, although limited, ability to introduce site-directed mutations during PCR.