Heterogeneous assay for pyrophosphate
AssigneeLI-COR, Inc.
InventorJohn G. K. Williams
Life of this patent
Estimating the term…
Abstract
Nucleotide triphosphate probes containing a fluorophore attached to the γ-phosphate and a quencher moiety sufficiently proximal to the fluorophore moiety for use in pyrophosphate detection assays are disclosed. These probes exhibit distinguishable fluorescence characteristics when the fluorophore is attached to the nucleotide through the γ-phosphate and when it is unattached to the nucleotide. The present invention also provides kits and integrated systems for practicing the assays described herein.
Claims25
A heterogeneous assay method for detecting pyrophosphate cleavage, the components of the assay comprising a labeled NTP, a target nucleic acid, a primer nucleic acid and a polymerase, said method comprising:
- (a) flowing said labeled nucleotide triphosphate (NTP) having a γ-phosphate with a fluorophore moiety attached thereto, past an immobilized component selected from the group consisting of said polymerase and said target nucleic acid;
- (b) incorporating said NTP on a primer strand hybridized to said target nucleic acid using said polymerase and releasing said γ-phosphate with said fluorophore moiety attached thereto;
- and
- (c) detecting said fluorescent moiety thereby detecting pyrophosphate cleavage.
An assay method for detecting pyrophosphate cleavage, the components of the assay comprising a labeled NTP, a target nucleic acid, a primer nucleic acid and a polymerase, said method comprising:
- (a) flowing said labeled nucleotide triphosphate (NTP) having a γ-phosphate with a fluorophore moiety attached thereto, past an immobilized component selected from the group consisting of said polymerase and said target nucleic acid;
- (b) incorporating said NTP on a primer strand hybridized to said target nucleic acid using said polymerase and releasing said γ-phosphate with said fluorophore moiety attached thereto;
- and
- (c) detecting said fluorescent moiety thereby detecting pyrophosphate cleavage.
A method for sequencing a target nucleic acid using an assay method for detecting pyrophosphate cleavage, the components of the assay comprising a labeled NTP, a target nucleic acid, a primer nucleic acid and a polymerase, said method comprising:
- (a) providing said labeled nucleotide triphosphate (NTP) having a γ-phosphate with a fluorophore moiety attached thereto, and an immobilized component selected from the group consisting of said polymerase and said target nucleic acid;
- (b) incorporating said NTP on said primer strand hybridized to said target nucleic acid using said polymerase and releasing said γ-phosphate with said fluorophore moiety attached thereto;
- and
- (c) detecting said fluorescent moiety by detecting pyrophosphate cleavage, thereby sequencing said target nucleic acid.
Dependencies are inferred from the claim text.