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Updated: Apr 9, 2026
DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
Published on: October 6, 2017
Measurement of DNA Polymerase Incorporation Kinetics of Dye-Labeled Nucleotides Using Total Internal Reflection
Matthew T Walsh1, Eric E Roller1, Kwang-Seuk Ko1
1Department of Bioengineering, University of California at San Diego, La Jolla, California 92093-0412, United States.
Abstract:
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.
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