An Internal Amplification Control System Based on Primer-Dimer Formation for PCR Product Detection by DNA Hybridization

Abstract

The detection of PCR products by DNA hybridization techniques can suffer from inhibition of the amplification process by sample matrix components. We have designed a simple internal control system for PCR based on the incorporation of a primer pair with complementary 3′ ends, resulting in the generation of a unique “primer-dimer” detectable by hybridization with a specific capture probe immobilized on polyester cloth as part of an array of amplicon-specific probes. The inclusion of this primer pair did not adversely affect the amplification and subsequent detection of target gene sequences by hybridization with immobilized probes in either single gene amplification or multiplex PCR systems. The failure to amplify target gene sequences because of the presence of inhibitors was mirrored by a failure to amplify the internal control primer-dimer, demonstrating the efficacy of this system in identifying the presence of DNA amplification inhibitors.