A quantitative fluorescence-based steady-state assay of DNA polymerase

Research output: Research - peer-reviewArticle

  • Authors:
  • Max D Driscoll
  • Julius Rentergent
  • Sam Hay

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.

Bibliographical metadata

Original languageEnglish
Pages (from-to)2042-2050
Number of pages9
JournalFEBS Journal
Volume281
Issue number8
DOIs
StatePublished - Apr 2014

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