Stopped-flow DNA polymerase assay by continuous monitoring of dNTP incorporation by fluorescence

DNA polymerase activity was measured by a stopped-flow assay that monitors polymerase extension using an intercalating dye. Double-stranded DNA formation during extension of a hairpin substrate was monitored at 75 °C for 2 min. Rates were determined in nucleotides per second per molecule of polymerase (nt/s) and were linear with time and polymerase concentration from 1 to 50 nM. The concentrations of 15 available polymerases were quantified and their extension rates determined in 50 mM Tris, pH 8.3, 0.5 mg/ml BSA, 2 mM MgCl₂, and 200 μM each dNTP as well as their commercially recommended buffers. Native Taq polymerases had similar extension rates of 10–45 nt/s. Three alternative polymerases showed faster speeds, including KOD (76 nt/s), Klentaq I (101 nt/s), and KAPA2G (155 nt/s). Fusion polymerases including Herculase II and Phusion were relatively slow (3–13 nt/s). The pH optimum for Klentaq extension was between 8.5 and 8.7 with no effect of Tris concentration. Activity was directly correlated to the MgCl₂ concentration and inversely correlated to the KCl concentration. This continuous assay is relevant to PCR and provides accurate measurement of polymerase activity using a defined template without the need of radiolabeled substrates.