Measurement of incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases using fluorescence microscopy.

We describe a method for measuring the single-turnover incorporation kinetics of non-fluorescent native nucleotides by DNA polymerases. Time-lapse total internal reflection fluorescence (TIRF) microscopy is used to directly measure the kinetics of single-base nucleotide incorporation into primed DNA templates covalently attached to the surface of a glass coverslip using a fixed ratio of a native nucleotide and a corresponding fluorescently labeled nucleotide over a series of total nucleotide concentrations. The presence of a labeled nucleotide allows for the kinetics of competitive incorporation reactions by DNA polymerase to be monitored. The single-turnover catalytic rate constants and Michaelis constants of the incorporation of the native nucleotides can be determined by modeling the kinetics of the parallel competitive reactions. Our method enables the measurements of the kinetics parameters of incorporation of native or other non-fluorescent nucleotides without using a rapid stopped-flow or quench-flow instrument and the generally more involved and less quantitative post-reaction analysis of the reaction products. As a demonstration of our method, we systematically determined the single-turnover incorporation kinetics of all four native nucleotides and a set of Cy3-labeled nucleotides by the Klenow fragment of Escherichia coli DNA polymerase I.