Comparison between Taq DNA Polymerase and Its Stoffel Fragment for Quantitative Real-Time PCR with Hybridization Probes

Abstract

In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan® probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in Light-Cycler® assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart® antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.