Primer Selection and Design for Polymerase Chain Reaction

One of the most important factors affecting the quality of polymerase chain reaction (PCR) is the choice of primers. Several rules should be observed when designing primers and, in general, the more DNA sequence information available, the better the chance of finding an “ideal” primer pair. Fortunately, not all primer selection criteria need be met in order to synthesize a clean, specific product, as the adjustment of PCR conditions (such as composition of the reaction mixture, temperature, and duration of PCR steps) may considerably improve the reaction specificity. Amplification of 200–400 bp DNA is the most efficient and, in these cases, one may design efficient primers simply by following a few simple rules described in this chapter. It is more difficult to choose primers for efficient amplification of longer DNA fragments, and use of an appropriate primer analysis software is worthwhile.