Journal
FEBS JOURNAL
Volume 281, Issue 8, Pages 2042-2050Publisher
WILEY
DOI: 10.1111/febs.12760
Keywords
DNA quantification; enzyme kinetics; fluorescence; Michaelis-Menten; PicoGreen; polymerase; steady-state
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Funding
- UK British Biological Sciences Research Council (BBSRC)
- BBSRC [BB/H021523/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/H021523/1, 1066097] Funding Source: researchfish
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Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the invitro 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 k(cat) and K-m values for the DNA polymerase that are in excellent agreement with literature values.
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