title: A quantitative fluorescence-based steady-state assay of DNA polymerase.
abstract: Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the in vitro measurement of the activity of DNA polymerases and nucleases. However, this approach is generally used in a semi-quantitative way to determine relative rates of reaction. In this report, we demonstrate a method for the simultaneous quantification of DNA in both its single-strand and double-strand forms using the dye PicoGreen. This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo(−), where we determine kcat and Km values for the DNA polymerase that are in excellent agreement with literature values.
authors.forename: Max D
authors.initials: MD
authors.lastname: Driscoll
authors.forename: Julius
authors.initials: J
authors.lastname: Rentergent
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
mesh_terms: D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase