Analyte detection
First Claim
1. A method of detecting an analyte comprising the steps of:
- (a) anchoring said analyte to a nucleic acid template;
(b) conducting a nucleic acid polymerase reaction to produce labeled polyphosphate, said reaction comprising the reaction of said template, a primer, at least one terminal phosphate-labeled nucleotide, and a nucleic acid polymerase; and
(c) analyzing said labeled polyphosphate.
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Accused Products
Abstract
A method of characterizing an analyte sample is provided that includes the steps of: (a) anchoring the analyte to a nucleic acid template of known sequence; (b) conducting a DNA polymerase reaction that includes the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide, DNA polymerase, and an enzyme having 3′→5′ exonuclease activity which reaction results in the production of labeled polyphosphate; (c) permitting the labeled polyphosphate to react with a phosphatase to produce a detectable species characteristic of the sample; (d) detecting the detectable species. The method may include the step of characterizing the nucleic acid sample based on the detection. Also provided are methods of analyzing multiple analytes in a sample, and kits for characterizing analyte samples.
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Citations
40 Claims
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1. A method of detecting an analyte comprising the steps of:
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(a) anchoring said analyte to a nucleic acid template;
(b) conducting a nucleic acid polymerase reaction to produce labeled polyphosphate, said reaction comprising the reaction of said template, a primer, at least one terminal phosphate-labeled nucleotide, and a nucleic acid polymerase; and
(c) analyzing said labeled polyphosphate. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A kit for detecting an analyte comprising:
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(a) at least one terminal-phosphate-labeled nucleotide;
(b) a DNA polymerase; and
(c) a phosphatase. - View Dependent Claims (32, 33, 34, 35)
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36. A method of detecting and characterizing multiple analytes in a sample, comprising the steps of:
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(a) anchoring to each analyte a specific template nucleic acid sequence with a unique base at the site opposite to the complementary nucleotide being added;
(b) conducting a DNA polymerase reaction to produce labeled polyphosphates, said reaction comprising the reaction of said templates, primers complementary to said specific template sequence, two or more terminal phosphate-labeled nucleotides with different labels, a DNA polymerase and an enzyme having 3′
→
5′
exonuclease activity;
(c) permitting said labeled polyphosphates to react with a phosphatase to produce detectable species unique to each of said analytes; and
(d) detecting said detectable species.
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37. A method of detecting and characterizing multiple analytes in a sample, comprising the steps of:
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(a) anchoring to each analyte a specific template nucleic acid sequence with a unique base at the site opposite to the complementary nucleotide being added;
(b) conducting a DNA polymerase reaction to produce uniquely labeled polyphosphates;
said reaction comprising the reaction of said templates, nuclease resistant primers complementary to said specific target sequence of each of said multiple analytes, two or more terminal phosphate-labeled nucleotides having 4 or more phosphate groups in the polyphosphate chain and each bearing a different label, a DNA polymerase and an enzyme having 3′
→
5′
exonuclease activity; and
(c) detecting the labeled polyphosphates.
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38. A method of detecting and characterizing multiple analytes in a reaction compartment, comprising the steps of:
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(a) anchoring a unique template nucleic acid sequence to each of said analytes;
(b) anchoring said analytes to the surface of said reaction compartment;
(c) conducting a DNA polymerase reaction to produce labeled polyphosphate;
said reaction comprising the reaction of the unique template sequence of one of said analytes, a nuclease resistant primer complementary to said unique template sequence, at least one terminal phosphate-labeled nucleotides having 4 or more phosphate groups in the polyphosphate chain, a DNA polymerase and an enzyme having 3′
→
5′
exonuclease activity;
(d) detecting said labeled polyphosphate;
(e) washing off the unanchored components; and
(f) repeating steps (a) to (d) with a nuclease resistant primer complementary to another unique template sequence of a different analyte until all the analytes are analyzed. - View Dependent Claims (39, 40)
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Specification