METHOD FOR DETECTING DNA POLYMERISATION

摘要

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.