ABSTRACT

The PCR essentially uses a DNA polymerase to increase the amount of a target DNA exponentially, doubling every “cycle” of the process. As Saiki (1989) notes “reduced to its most basic terms, PCR merely involves combining a DNA sample with oligonucleotide primers, deoxynucleotide triphosphates, and the thermostable Taq DNA polymerase in a suitable buffer, then repetitively heating and cooling the mixture for several hours until the desired amount of amplification is achieved.” However, all too common experiences with the PCR include problems such as mispriming at non-target sites, primer dimer formation, failed denaturation, mutant PCR product, and contamination. Thus, Saiki (1989) goes on to observe that “In fact, the PCR is a relatively complicated and, as yet, incompletely understood biochemical brew where constantly changing kinetic interactions among the several components determine the quality of the products obtained.” It is the goal of this chapter to enable the reader to understand that biochemical brew well enough to achieve the best possible PCR results.