title: Measurement of DNA Polymerase Incorporation Kinetics of Dye-Labeled Nucleotides Using Total Internal Reflection Fluorescence Microscopy.
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.
authors.affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, California 92093-0412, United States.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Walsh
authors.affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, California 92093-0412, United States.
authors.forename: Eric E
authors.initials: EE
authors.lastname: Roller
authors.affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, California 92093-0412, United States.
authors.forename: Kwang-Seuk
authors.initials: KS
authors.lastname: Ko
authors.affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, California 92093-0412, United States.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Huang
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D009711:Nucleotides / Q000032:analysis / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D009711:Nucleotides; D004259:DNA-Directed DNA Polymerase