Method for detecting dna polymerisation
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United States of America Patent
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Issued Date -
Sep 23, 2004
app pub date -
Nov 14, 2003
filing date -
Feb 14, 2001
priority date (Note) -
Abandoned
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Abstract
A method for determining the extent of a processive nucleic acid polymerase reaction producing pyrophosphate can be conducted, in the presence of all components necessary for the progression of nucleic acid synthesis, wherein the components comprise a substrate for the nucleic acid polymerase which is either dATP or a dATP analogue. The method includes a pyrophosphate assay comprising the steps of conversion of the pyrophosphate to ATP and detection of light produced by the bioluminescence reaction of a luciferase with ATP, wherein one or both of the following apply: a) the luciferase reacts with ATP and the substrate, such that the spectral overlap is reduced relative to the spectral overlap between the outputs of reaction between wild-type Photinus pyralis luciferase with ATP and dATP respectively; and b) the luciferase reacts with the substrate to give a reduced bioluminescence relative to that produced by the reaction of wild-type Photinus pyralis luciferase with dATP.
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