Single nucleotide amplification and detection by polymerase

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.

2. The method of claim 1 wherein step (a) further comprises conducting said polymerase reaction in the presence of a phosphatase.

3. The method of claim 1 wherein said detectable species is produced in amounts substantially proportional to the amount of nucleic acid sample.

4. The method of claim 1 further comprising the step of quantifying said nucleic acid sample.

5. The method of claim 1 wherein said nucleic acid sample comprises DNA or RNA.

6. The method of claim 1 wherein step (a) further comprises conducting said polymerase reaction in the presence of two or more terminal phosphate-labeled nucleotides with distinct labels.

7. The method of claim 6 wherein said labels are enzyme-activatable labels selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

8. The method of claim 1 wherein said terminal-phosphate-labeled nucleotide may be represented by formula I:

wherein P=phosphate (PO 3 ) and, n is 2 or greater; Y is an oxygen or sulfur atom; B is a nitrogen-containing heterocyclic base; S is an acyclic moiety, carbocyclic moiety or sugar moiety; L is an enzyme-activatable label containing a hydroxyl group, a sulfhydryl group or an amino group suitable for forming a phosphate ester, a thioester or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide; P-L is a phosphorylated label which preferably becomes independently detectable when the phosphate is removed.

9. The method of claim 1 , wherein said terminal phosphate-labeled nucleotide comprises four or more phosphate groups in the polyphosphate chain.

10. The method of claim 1 further comprising the step of including one or more additional detection reagents in said polymerase reaction.

11. The method of claim 10 wherein said additional detection reagents are capable of a response that is detectably different from said detectable species.

12. The method of claim 8 wherein said enzyme-activatable label is selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

13. The method of claim 1 wherein said detectable species is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, mass change, oxidation/reduction potential and combinations thereof.

14. The method of claim 12 wherein said phosphorylated label is a fluorogenic moiety selected from the group consisting of 2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone, fluorescein diphosphate, fluorescein 3′(6′)-O-alkyl-6′(3′)-phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate, 4-methylumbelliferyl phosphate, resorufin phosphate, 4-trifluoromethylumbelliferylphosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate and 6,8-difluoro-4-methylumbelliferyl phosphate.

15. The method of claim 12 wherein said phosphorylated label is a chromogenic moiety selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate.

16. The method of claim 12 wherein said chemiluminescent compound is an alkaline phosphatase-activated 1,2-dioxetane compound.

17. The method of claim 16 wherein said 1,2-dioxetane compound is selected from the group consisting of 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2-(5-chloro-)tricycle[3,3,1-1 3,7 ]-decan]-1-yl)-1-phenyl phosphate, chloroadamant-2′-ylidenemethoxyphenoxyphosphorylated dioxetane, 3-(2′-spiroadamrtre)-4-methoxy-4-(3″-phosphovloxy)phenyl-1,2-dioxetane.

18. The method of claim 8 wherein said sugar moiety is selected from the group consisting of ribosyl, 2′-deoxyribosyl, 3′-deoxyribosyl, 2′,3′-dideoxyribosyl, 2′,3′-didehydrodideoxyribosyl, 2′-alkoxyribosyl, 2′-azidoribosyl, 2′-aminoribosyl, 2′-fluororibosyl, 2′-mercaptoriboxyl, 2′-alkylthioribosyl, carbocyclic, acyclic and other modified sugars.

19. The method of claim 8 wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, 7-deazaguanine, hypoxanthine, 7-deazahypoxanthine, adenine, 7-deazaadenine, 2,6-diaminopurine.

20. The method of claim 1 wherein said non-hydrolyzable primer is a methyl phosphonate, a borano phosphate or is phosphorothioated at the 3′-most phosphodiester linkage.

21. The method of claim 1 wherein said DNA polymerase is selected from the group consisting of the Klenow fragment of DNA polymerase I, Phi 29 DNA polymerase, DNA polymerase I, T4 DNA polymerase, Thermo Sequenase, Amplitaq FS, reverse transcriptase and T7 DNA polymerase.

22. 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.

23. The method of claim 22 wherein step (a) further comprises conducting said polymerase reaction in the presence of a phosphatase.

24. The method of claim 22 wherein said detectable species is produced in amounts substantially proportional to the amount of nucleic acid sample.

25. The method of claim 22 further comprising the step of quantifying said nucleic acid sample.

26. The method of claim 22 wherein said nucleic acid sample comprises DNA or RNA.

27. The method of claim 22 wherein step (a) further comprises conducting said polymerase reaction in the presence of two or more terminal phosphated-labeled nucleotides with distinct labels.

28. The method of claim 27 wherein said labels are enzyme-activatable labels selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

29. The method of claim 22 wherein said terminal-phosphate-labeled nucleotide may be represented by formula I:

wherein P=phosphate (PO 3 ) and, n is 2 or greater; Y is an oxygen or sulfur atom; B is a nitrogen-containing heterocyclic base; S is an acyclic moiety, carbocyclic moiety or sugar moiety; L is an enzyme-activatable label containing a hydroxyl group, a sulfhydryl group or an amino group suitable for forming a phosphate ester, a thioester or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide; P-L is a phosphorylated label which preferably becomes independently detectable when the phosphate is removed.

30. The method of claim 22 , wherein said terminal phosphate-labeled nucleotide comprises four or more phosphate groups in the polyphosphate chain.

31. The method of claim 22 further comprising the step of including one or more additional detection reagents in said polymerase reaction.

32. The method of claim 31 wherein said additional detection reagents are capable of a response that is detectably different from said detectable species.

33. The method of claim 29 wherein said enzyme-activatable label is selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags, and combinations thereof.

34. The method of claim 22 wherein said detectable species is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, mass change, oxidation/reduction potential and combinations thereof.

35. The method of claim 33 wherein said phosphorylated label is a fluorogenic moiety selected from the group consisting of 2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone, fluorescein diphosphate, fluorescein 3′(6′)-O-alkyl-6′(3′)-phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate, 4-methylumbelliferyl phosphate, resorufin phosphate, 4 -trifluoromethylumbelliferylphosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate.

36. The method of claim 33 wherein said phosphorylated label is a chromogenic moiety selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate.

37. The method of claim 33 wherein said chemiluminescent compound is a phosphatase-activated 1,2-dioxetane compound.

38. The method of claim 37 wherein said 1,2-dioxetane compound is selected from the group consisting of 2-chloro-5-(4-meloxyspiro[1,2-dioxetane-3,2-(5-chloro-)tricycle[3,3,1-1 3,7 ]-decan]-1-yl)-1-phenyl phosphate, chloroadamant-2′-ylidenemethoxyphenoxyphosphorylated dioxetane and 3-(2′-spiroadamrtre)-4-methoxy-4-(3″-phosphovloxy)phenyl-1,2-dioxetane.

39. The method of claim 29 wherein said sugar moiety is selected from the group consisting of ribosyl, 2′-deoxyribosyl, 3′-deoxyribosyl, 2′,3′-dideoxyribosyl, 2′,3′-didehydrodideoxy-ribosyl, 2′-alkoxyribosyl, 2′-azidoribosyl, 2′-aminoribosyl, 2′-fluororibosyl, 2′mercaptoriboxyl, 2′-alkylthioribosyl, carbocyclic, acyclic and other modified sugars.

40. The method of claim 29 wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, 7-deazaguanine, hypoxanthine, 7-deazahypoxanthine, adenine, 7 -deazaadenine, 2,6-diaminopurine.

41. The method of claim 22 wherein said non-hydrolyzable primer is a methyl phosphonate, a borano phosphate or is phosphorothioated at the 3′-most phosphodiester linkage.

42. The method of claim 22 wherein said DNA polymerase is selected from the group consisting of the Klenow fragment of DNA polymerase I, Phi 29 DNA polymerase, DNA polymerase I, T4 DNA polymerase, Thermo Sequenase, Amplitaq FS, reverse transcriptase and T7 DNA polymerase.

43. 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.

44. 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.

45. 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.

46. 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.