Single nucleotide amplification and detection by polymerase
AssigneeAmersham Biosciences AB
InventorsJohn Nelson, Carl Fuller, Anup Sood, Shiv Kumar
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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.
Claims46
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.
A method of detecting 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;
- and
- (c) detecting said detectable species.
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 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.
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 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.
A method of detecting 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 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.
A method of detecting 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 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.
Dependencies are inferred from the claim text.