Methods in Molecular Biology PCR Protocols SECOND EDITION

1. Introduction The polymerase chain reaction (PCR) is a powerful method for fast in vitro enzymatic amplifications of specific DNA sequences. PCR amplifications can be grouped into three different categories: standard PCR, long PCR, and multiplex PCR. Standard PCR involves amplification of a single DNA sequence that is less than 5 kb in length and is useful for a variety of applications, such as cycle sequencing, cloning, mutation detection, etc. Long PCR is used for the amplification of a single sequence that is longer than 5 kb and up to 40 kb in length. Its applications include long-range sequencing; amplification of complete genes; PCR-based detection and diagnosis of medically important large-gene insertions or deletions; molecular cloning; and assembly and production of larger recombinant constructions for PCR-based mutagenesis (1,2). The third category, multiplex PCR, is used for the amplification of multiple sequences that are less than 5 kb in length. Its applications include forensic studies; pathogen identification; linkage analysis; template quantitation; genetic disease diagnosis; and population genetics (3–5). Unfortunately, there is no single set of conditions that is optimal for all PCR. Therefore, each PCR is likely to require specific optimization for the template/primer pairs chosen. Lack of optimization often results in problems, such as no detectable PCR product or low efficiency amplification of the chosen template; the presence of nonspecific bands or smeary background; the formation of " primer-dimers " that compete with the chosen template/primer set for amplification; or mutations caused by errors in nucleotide incorporation. It is particularly important to optimize PCR that will be used for repetitive diagnostic or analytical procedures where optimal amplification is required. The objective of this chapter is to discuss the parameters that may affect the specificity, fidelity, and efficiency of PCR, as well as approaches that can be taken to achieve optimal PCR amplifications. Optimization of a particular PCR can be time consuming and complicated because of the various parameters that are involved. These parameters include the following: (1) quality and concentration of DNA template; (2) design and concentration of primers; (3) concentration of magnesium ions; (4) concentration of the four deoxynucleotides (dNTPs); (5) PCR buffer systems; (6) selection and concentration of DNA poly-merase; (7) PCR thermal cycling conditions; (8) addition and concentrations of PCR additives/cosolvents; and (9) use of the " hot start " technique. Optimization of PCR