Method to detect activity of a polymerase
AssigneesMax-Planck-Gesellschaft zur Forderung der Wissenschaften e.V., Lead Discovery Center GmbH
InventorsTim Bergbrede, Nils-Göran Larsson, Claes Gustafsson, Maria Falkenberg-Gustafsson
Life of this patent
Estimating the term…
Abstract
The present invention relates to methods for detection of nucleotide polymerase activity and methods of detecting compounds that modulate nucleotide polymerase activity, by detecting product formation of the nucleotide polymerase to be tested based on determination of close proximity of two labeled nucleotide probes able to bind the product of the nucleotide polymerase. It is preferred that proximity dependent energy transfer, such as forster resonance energy transfer, between said labeled nucleotide probes is determined. The invention further provides kits comprising components for carrying out the inventive methods for detection of nucleotide polymerase activity.
Claims23
A method for detection of nucleotide polymerase activity comprising the following steps:
- a) providing a reaction mixture comprising at least said polymerase, an initiator, and nucleoside triphosphates,
- b) providing conditions sufficient to allow the polymerase an assembling of nucleotides,
- c) stopping polymerase assembling of nucleotides,
- d) after the step of stopping polymerase assembling of nucleotides, adding a donor nucleotide probe and an acceptor nucleotide probe wherein said donor nucleotide probe is conjugated to a first non-radioactive label and said acceptor nucleotide probe is conjugated to a second non-radioactive label, wherein said labels allow determination of close proximity of said nucleotide probes,
- e) providing conditions sufficient to allow the nucleotide probes to hybridize with the product of step b) and
- f) determining if said donor and acceptor nucleotide probes are in close proximity wherein close proximity correlates with the activity of the nucleotide polymerase
- wherein said initiator is a template oligonucleotide or a primer for a template-independent nucleotide polymerase, and
- wherein said donor nucleotide probe and said acceptor nucleotide probe are complementary to the product of the nucleotide polymerase assembled at step b).
A method for detection of nucleotide polymerase activity comprising the following steps:
- a) providing a reaction mixture comprising said polymerase, an initiator, and nucleoside triphosphates,
- b) providing conditions sufficient to allow the polymerase an assembling of nucleotides,
- c) stopping polymerase assembling of nucleotides,
- d) after the step of stopping polymerase assembling of nucleotides, adding a donor nucleotide probe and an acceptor nucleotide probe wherein the donor nucleotide probe is conjugated to a fluorophore being a donor for förster resonance energy transfer and the acceptor nucleotide probe is conjugated to a fluorophore being a fluorescence quencher or an appropriate acceptor for förster resonance energy transfer,
- e) providing conditions sufficient to allow the nucleotide probes to hybridize with the product of step b) and
- f) determining fluorescence wherein said fluorescence correlates with the activity of the nucleotide polymerase;
- wherein said initiator is a template oligonucleotide or a primer for a template-independent nucleotide polymerase, and
- wherein said donor nucleotide probe and said acceptor nucleotide probe are complementary to the product of the nucleotide polymerase assembled at step b).
Dependencies are inferred from the claim text.