Protocol

Molecular Biotechnology

, Volume 3, Issue 2, pp 129-134

Selection of primers for polymerase chain reaction

  • Wojciech RychlikAffiliated withNational Biosciences Inc.

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Abstract

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowT m of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.

Index entries

PCR primer design primer internal stability