Mechanism and application of Taq DNA polymerase in TaqMan qPCR

TaqMan quantitative real-time PCR (qPCR) is widely used in molecular biology due to its high specificity, sensitivity, and multiplexing capability. Taq DNA polymerase (Taq pol) plays a central role by simultaneously catalyzing DNA synthesis and cleaving fluorogenic probes, yet the mechanistic interplay between these activities remains incompletely characterized. We systematically quantified Taq pol nuclease and polymerase activities during extension and probe cleavage. A novel screening assay integrating both extension and cleavage readouts was developed to accurately evaluate Taq pol performance. Several engineered Taq variants and KlenTaq (lacking the 5′-3′ exonuclease domain) were subsequently analyzed. Our analysis revealed a synergistic relationship between polymerase extension and probe cleavage activities. Two Taq variants exhibited significantly enhanced probe cleavage efficiency, while KlenTaq demonstrated markedly stronger strand displacement activity. These findings elucidate the molecular mechanisms governing Taq pol activity during qPCR. The identified variants with improved cleavage efficiency are promising for multiplex detection, whereas KlenTaq’s enhanced strand displacement supports specialized applications such as probe melting curve analysis. This work provides both theoretical insights and practical tools to optimize qPCR assays and engineer next-generation polymerases.