Single nucleotide amplification and detection by polymerase
First Claim
Patent Images
1. A method of characterizing a nucleic acid sample comprising the steps of:
- (a) conducting a DNA polymerase reaction, said reaction comprising 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, wherein said enzyme may be selected from the group consisting of DNA polymerases, exonucleases and combinations thereof, which reaction results in the production of labeled polyphosphate;
(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species characteristic of the sample;
(c) detecting said detectable species; and
(d) characterizing said nucleic acid sample based on said detection.
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Abstract
A method of characterizing a nucleic acid sample is provided that includes the steps of: (a) 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; (b) permitting the labeled polyphosphate to react with a phosphatase to produce a detectable species characteristic of the sample; (c) detecting the detectable species; and (d) characterizing the nucleic acid sample based on the detection.
186 Citations
46 Claims
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1. A method of characterizing a nucleic acid sample comprising the steps of:
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(a) conducting a DNA polymerase reaction, said reaction comprising 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, wherein said enzyme may be selected from the group consisting of DNA polymerases, exonucleases and combinations thereof, which reaction results in the production of labeled polyphosphate;(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species characteristic of the sample; (c) detecting said detectable species; and (d) characterizing said nucleic acid sample based on said detection. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method of detecting a nucleic acid sample comprising the steps of:
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(a) conducting a DNA polymerase reaction, said reaction comprising 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, wherein said enzyme may be selected from the group consisting of DNA polymerases, exonucleases and combinations thereof, which reaction results in the production of labeled polyphosphate;(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and (c) detecting said detectable species. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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43. A method of characterizing a nucleic acid sample comprising the steps of:
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(a) conducting a DNA polymerase reaction, said reaction comprising the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide having four or more phosphate groups in the polyphosphate chain, DNA polymerase and an enzyme having 3′
→
5′
exonuclease activity, wherein said enzyme may be selected from the group consisting of DNA polymerases, exonucleases and combinations thereof, which reaction results in the production of labeled polyphosphate;(b) detecting said labeled polyphosphate; and (c) characterizing the nucleic acid sample based on said detection.
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44. A method of characterizing a nucleic acid sample comprising the steps of:
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(a) conducting a DNA polymerase reaction, said reaction comprising the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide having four or more phosphate groups in the polyphosphate chain, DNA polymerase and an enzyme having 3′
→
5′
exonuclease activity, wherein said enzyme may be selected from the group consisting of DNA polymerases, exonucleases and combinations thereof, which reaction results in the production of labeled polyphosphate;(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species having a signal profile characteristic of the sample; (c) detecting said detectable species; and (d) characterizing said nucleic acid sample based on said signal profile.
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45. A method of detecting a nucleic acid sample comprising the steps of:
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(a) conducting a DNA polymerase reaction, said reaction comprising the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide having four or more phosphate groups in the polyphosphate chain, DNA polymerase and an enzyme having 3′
→
5′
exonuclease activity, wherein said enzyme may be selected from the group consisting of DNA polymerases, exonucleases and combinations thereof, which reaction results in the production of labeled polyphosphate; and(b) detecting said labeled polyphosphate.
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46. A method of detecting a nucleic acid sample comprising the steps of:
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(a) conducting a DNA polymerase reaction, said reaction comprising the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide having four or more phosphate groups in the polyphosphate chain, DNA polymerase and an enzyme having 3′
→
5′
exonuclease activity, wherein said enzyme may be selected from the group consisting of DNA polymerases, exonucleases and combinations thereof, which reaction results in the production of labeled polyphosphate;(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and (c) detecting said detectable species.
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Specification