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A quantitative fluorescence‐based steady‐state assay of DNA polymerase
Author(s) -
Driscoll Max D.,
Rentergent Julius,
Hay Sam
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12760
Subject(s) - klenow fragment , dna polymerase , polymerase , dna , dna polymerase i , microbiology and biotechnology , fluorescence , primer dimer , chemistry , real time polymerase chain reaction , dna clamp , polymerase chain reaction , biology , biophysics , biochemistry , reverse transcriptase , exonuclease , multiplex polymerase chain reaction , gene , physics , quantum mechanics
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 P ico G reen. This approach is used in a steady‐state assay of DNA polymerase K lenow fragment exo − , where we determine k cat and K m values for the DNA polymerase that are in excellent agreement with literature values.