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PCR Primer Design

Adapted from PCR Primer: A Laboratory Manual, 2nd edition (eds. Dieffenbach and Dveksler). CSHL Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

The objective of a polymerase chain reaction (PCR) is to amplify a specific DNA segment without any nonspecific by-products. In principle, each physical and chemical component of PCR can be modified to produce a potential increase in yield, specificity, or sensitivity. Yet the most critical parameter for successful PCR is optimal primer design. A poorly designed primer can result in little or no product, due to nonspecific amplification and/or primer-dimer formation leading to reaction failure, even when all the other parameters are properly optimized. This article provides general guidelines for PCR primer design, tips for development of primer pairs for more complex applications, and advice on the development of probes for real-time PCR.