Measurement of DNA Polymerase Incorporation Kinetics of Dye-Labeled Nucleotides Using Total Internal Reflection Fluorescence Microscopy

We report a method for the rapid and automated measurements of the incorporation kinetics of fluorescent dye-labeled nucleotides by DNA polymerases without using stopped-flow and quench-flow methods. Total internal reflection fluorescence microscopy is used to monitor the incorporation of fluorescently labeled nucleotides by DNA polymerase into surface-bound primed DNA templates, and a microfluidic system is used to perform the reactions. We successfully demonstrated the method using Bst DNA polymerase and a set of coumarin-labeled nucleotides. Our method allows the rapid acquisition of polymerase kinetics for implementing and improving DNA sequencing technologies that rely on labeled nucleotides and DNA polymerases.