Optimisation of the polymerase chain reaction

The polymerase chain reaction (PCR) is a method by which specific sequences of DNA can be copied many times, allowing detailed molecular studies to be performed on as little as a single cell. Numerous and diverse applications of PCR are being developed across all disciplines of diagnostic pathology and research, and no single protocol is appropriate for all situations. Optimising PCR requires a delicate balance between the amplification of specific products and avoiding the production of non-specific products. Each step, from DNA template extraction to cycling times and temperatures, needs to be considered carefully. The aim of this study is to assess which parameters influence DNA amplification efficiency and specificity. The parameters evaluated are the denaturation, annealing and extension temperatures, the number of cycles performed, and the primer, magnesium chloride, dNTP, Taq DNA polymerase and DNA template concentrations. The important parameters for efficient, specific amplification were denaturation time and temperature, stringent annealing temperatures and magnesium chloride concentration. The importance of DNA concentration was found to depend upon the source from which the DNA was extracted.