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OLIGO Primer Analysis Software

2003; Humana Press; Linguagem: Inglês

10.1007/978-1-59259-335-4_21

John D. Offerman, Wojciech Rychlik,

Genomics and Phylogenetic Studies

Concurrent with the development of the polymerase chain reaction (PCR) in the late 1980s as an essential technique in the molecular biology laboratory, optimal design of PCR primers became an important part of the process. It was soon learned, for example, that inattention to secondary structure in synthetic primers could create primer-dimers and hairpins and could seriously reduce or eliminate the yield of the PCR product. The presence of priming sites in the template other than the intended target—so called false priming sites—were also found to interfere with PCR efficiency, by generating unwanted PCR products and background on the gel. Furthermore, the efficient PCR experiment required the proper concentrations of salt, buffer, and nucleic acid and the accurate determination of melting temperatures of the primers and the template. Cumbersome calculations were required to produce these values and their complexity often resulted in errors.