Troubleshooting fine-tuning procedures for qPCR system design

Abstract

Quantitative real-time PCR (qPCR) has been improved and optimized over the past decade for a wide range of applications. Design of primers and probes is one of the crucial steps to obtain high system efficiency of qPCR since design pitfalls influence negatively amplification performances. We report the results of some experiments. First, we demonstrate the utility of optimal primer design and concentration in PCR by constructing suboptimal primers, for instance with hairpin and primer-dimers secondarystructures, and quantifying the decrease in efficiency of amplification. Second, we show the adverse effects of the target sequence harboring stable secondary structures on the primer binding sites. Finally, we let see that the mere use of probe-based detection is not enough to ensure robustness of qPCR data, because the eventual detrimental products generated by primers not well designed may influence in any case the PCR efficiency. © 2011 Wiley Periodicals, Inc.