A quantitative fluorescence‐based steady‐state assay of DNA polymerase. (10th March 2014)

• Record Type: Journal Article
• Authors: Driscoll, Max D.; Rentergent, Julius; Hay, Sam
• Is Part Of: FEBS journal. Volume 281:Number 8(2014)
• Page Start: 2042
• Page End: 2050
• Publication Date: 2014-03-10
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.12760
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs12760-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <p>Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the <italic>in vitro</italic> 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 <italic>k</italic>_cat and <italic>K</italic>_m values for the DNA polymerase that are in excellent agreement with literature values.</p> </abstract>