A guide to the design and use of mismatched and degenerate primers.
Roche Molecular Systems, Inc., Alameda, California 94501 Although simple in concept, PCR requires a myriad of complex interactions between template, primers, deoxynucleoside triphosphates, and DNA polymerase to successfully accomplish targeted amplification. Primer design in conjunct ion with changes in the concentrat ions of reaction components and thermal cycling parameters provides a versatility available in no other molecular technique. For example, PCR can be employed to accommodate mismatches in the pr imertemplate duplex, thereby permit t ing amplification not only of related sequences but also of uncharacterized sequences. It can also be designed to amplify selectively a small number of mutan t genes with single base alterations in a vast background of normal genes. The flexibiliW of PCR has greatly simplified molecular manipulat ions. Procedures for altering a particular template sequence, which in the past required several steps, can now be performed with only a few manipulat ions. Because 3' and internal mismatches between pr imertemplate duplexes are tolerated under appropriate conditions, point mutat ions and desired restriction endonuclease sites can be directly introduced into the primers. Likewise, nucleotide insertions and deletions can be similarly introduced into the amplified product via the primers. Degenerate primers have made it possible to amplify related but distinct nucleic acid sequences as well as to amplify targets for which only amino acid sequences are available. The range of possible applications precludes providing detailed protocols for each. Instead, the delineation of the critical parameters and a guide for representative use will be summarized. Depending on the application, a subset of these recommendat ions or suggestions may suffice.