Specificity, efficiency, and fidelity of PCR.

Center for Environmental Health Sciences and Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 The efficacy of PCR is measured by its specificity, efficiency (i.e. yield), and fidelity. A highly specific PCR will generate one and only one amplification product that is the intended target sequence. More efficient amplification will generate more products with fewer cycles. A highly accurate (i.e., high-fidelity) PCR, will contain a negligible amount of DNA polymerase-induced errors in its product. An ideal PCR would be the one with high specificity, yield, and fidelity. Studies indicate that each of these three parameters is influenced by numerous components of PCR, including the buffer conditions, the PCR cycling regime (i.e., temperature and duration of each step), and DNA polymerases. Unfortunately, adjusting conditions for maximum specificity may not be compatible with high yield; likewise, optimizing for the fidelity of PCR may result in reduced efficiency. Thus, when setting up a PCR, one should know which of the three parameters is the most important for its intended application and optimize PCR accordingly. For instance, for direct sequencing analysis of a homogenous population of ceils (either by sequencing or by RFLP), the yield and specificity of PCR is more important than the fidelity. On the other hand, for studies of individual DNA molecules, or rare mutants in a heterogeneous population, fidelity of PCR is vital. The purpose of current communication is to focus on the essential components of setting up an effective PCR, and discuss how each of these component may influence the specificity, efficiency, and fidelity of PCR.

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