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

ABSTRACT: 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 ki

SUBMITTER: Walsh MT 

PROVIDER: S-EPMC5716174 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature