Terminal-phosphate-labeled nucleotides and methods of use

• US 7,052,839 B2
• Filed: 04/01/2002
• Issued: 05/30/2006
• Est. Priority Date: 08/29/2001
• Status: Expired due to Term

• Current Assignee: Global Life Sciences Solutions USA LLC (Danaher Corporation)
• Original Assignee: Amersham Biosciences Corp. (General Electric Company)
• Inventors: Nelson, John, Sood, Anup, Kumar, Shiv, Fuller, Carl
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Calamita, Heather G.
• Application Number:
  US10/113,030
• Publication Number:
  US 20030077610A1
• Time in Patent Office: 1,520 Days
• Field of Search: 435/6, 435/7, 435/91.2, 435/7.1, 436/501
• US Class Current: 435/5
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: C09B 19/00 Oxazine dyes
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C09B 69/109 containing other specific dyes
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification

Abstract

The present invention describes methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases. The methods provided by this invention utilize a nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent moiety, a mass tag or an electrochemical tag attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Cleavage of the polyphosphate product of phosphoryl transfer via phosphatase leads to a detectable change in the label attached thereon. When the polymerase assay is performed in the presence of a phosphatase, there is provided a convenient method for real-time monitoring of DNA or RNA synthesis and detection of a target nucleic acid.

Claims

1. A method of detecting the presence of a nucleic acid sequence comprising the steps of:

a) conducting a nucleic acid polymerase reaction, said reaction comprising the reaction of at least one nucleotide which is substantially non-reactive to phosphatase and at least one terminal-phosphate-labeled nucleotide, 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 the presence of said detectable species.

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 nucleic acid sequence is RNA.

4. 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.

5. The method of claim 4 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.

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

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

8. The method of claim 1 further comprising the step of quantifying said nucleic acid sequence.

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

10. The method of claim 1 wherein said nucleic acid sequence is a natural or synthetic oligonucleotide.

11. The method of claim 1 wherein said nucleic acid sequence is a chromosome or part of a chromosome.

12. The method of claim 1 wherein said nucleic acid sequence is DNA.

13. The method of claim 1 wherein said polymerase reaction further comprises the step of incubating a nucleic acid sequence in the presence of at least one of DNA or RNA polymerase.

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

15. The method of claim 14 wherein said additional detection reagents are capable of a response that is detectably different from said detectable species.

16. The method of claim 14 wherein said additional detection reagent is an antibody.

17. The method of claim 6 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.

18. 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, reduction/oxidation potential and combinations thereof.

19. The method of claim 6 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, resorutin phosphate, 4-triiuoromethylumbelliferylphosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate and 6,8-difluoro-4-methylumbelliferyl phosphate.

20. The method of claim 6 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 and derivatives thereof.

21. The method of claim 6 wherein said chemiluminescent compound is a phosphatase-activated 1,2-dioxetane compound.

22. The method of claim 21 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)-1phenyl phosphate, chloroadamant-2′

-ylidenemethoxyphenoxyphosphorylated dioxetane and 3-(2′

-spiroadamrtre)-4-methoxy-4-(3″

-phosphovloxy)phenyl-1,2-dioxetane.

23. The method of claim 6 wherein said sugar moiety is selected from the group consisting 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.

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

25. The method of claim 1 further comprising the step of quantifying said nucleic acid sequence by comparison of spectra produced by said detectable species with known spectra.

26. A method of detecting the presence of a DNA sequence comprising the steps of:

a) conducting a DNA polymerase reaction in the presence of a terminal-phosphate-labeled nucleotide, which reaction results in the production of a labeled polyphosphate;

b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and

c) detecting the presence of said detectable species.

27. The method of claim 26 wherein step (a) further comprises conducting said polymerase reaction in the presence of a phosphatase.

28. The method of claim 26 wherein step (a) further comprises conducting said polymerase reaction in the presence of two or more terminal-phosphate-labeled nucleotides with distinct labels.

29. The method of claim 28 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.

30. The method of claim 26 wherein said terminal-phosphate-labeled nucleotide may be represented by formula I:

31. The method of claim 26, wherein said terminal phosphate-labeled nucleotide comprises four or more phosphate groups in the polyphosphate chain.

32. The method of claim 26 further comprising the step of quantifying said DNA sequence.

33. The method of claim 26 wherein said detectable species is produced in amounts substantially proportional to the amount of sequence.

34. The method of claim 26 wherein said DNA sequence is a natural or synthetic oligonucleotide.

35. The method of claim 26 wherein said DNA sequence is a chromosome or part of a chromosome.

36. The method of claim 26 wherein said polymerase reaction further comprises the step of incubating a DNA sequence in the presence of a DNA polymerase.

37. The method of claim 26 further comprising the step of including one or more additional detection reagents in said polymerase reaction.

38. The method of claim 37 wherein said additional detection reagents are capable of a response that is detectably different from said detectable species.

39. The method of claim 37 wherein said additional detection reagent is an antibody.

40. The method of claim 30 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.

41. The method of claim 26 wherein said detectable species is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, mass change, reduction/oxidation potential and combinations thereof.

42. The method of claim 30 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, resorutin phosphate, 4-triiuoromethylumbelliferylphosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate and 6,8-difluoro-4-methylumbelliferyl phosphate.

43. The method of claim 30 wherein said phosphorylated label is a chromogenic moiety selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate and p-nitrophenyl phosphate.

44. The method of claim 30 wherein said chemiluminescent compound is a phosphatase-activated 1,2-dioxetane compound.

45. The method of claim 44 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.

46. The method of claim 30 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.

47. The method of claim 30 wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, 7-deazaguanine, hypoxantinine, 7-deazahypoxanthine, adenine, 7-deazaadenine and 2,6-diaminopurine.

48. The method of claim 26 further comprising the step of quantifying said DNA sequence by comparison of spectra produced by said detectable species with known spectra.

49. A method of quantifying a nucleic acid comprising the steps of:

(a) conducting a nucleic acid polymerase reaction, said reaction comprising the reaction of nucleotide which is substantially non-reactive to phosphatase and at least one terminal-phosphate-labeled nucleotide;

said reaction resulting in production of labeled polyphosphate;

(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable by-product species in amount substantially proportional to the amount of said nucleic acid;

(c) measuring said detectable species; and

(d) comparing said measurements using known standards to determine the quantity of nucleic acid.

50. The method of claim 49 wherein step (a) further comprises conducting said polymerase reaction in the presence of a phosphatase.

51. The method of claim 49 wherein step (a) further comprises conducting said polymerase reaction in the presence of two or more terminal-phosphate-labeled nucleotides with distinct labels.

52. The method of claim 51 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.

53. The method of claim 49 wherein said terminal-phosphate-labeled nucleotide may be represented by formula I:

54. The method of claim 53, wherein said terminal phosphate-labeled nucleotide comprises four or more phosphoryl groups in the phosphate chain.

55. The method of claim 53 wherein said phosphatase changes said phosphate ester linkage to produce said detectable species.

56. The method of claim 53 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.

57. The method of claim 53 wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, 7-deazaguanine, hypoxantinine, 7-deazahypoxanthine, adenine, 7-deazaadenine and 2,6-diaminopurine.

58. The method of claim 53 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.

59. The method of claim 53 wherein detectable species is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, mass change, reduction/oxidation potential and combinations thereof.

60. A method of quantifying a DNA sequence comprising the steps of:

(a) conducting a DNA polymerase reaction in the presence of a terminal-phosphate-labeled nucleotide, said reaction resulting in production of labeled polyphosphate;

(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable by-product species in amounts substantially proportioned to the amount of said DNA sequence;

(c) measuring said detectable species; and

(d) comparing said measurements using known standards to determine the quantity of DNA.

61. The method of claim 60 wherein step (a) further comprises conducting said polymerase reaction in the presence of a phosphatase.

62. The method of claim 60 wherein step (a) further comprises conducting said polymerase reaction in the presence of two or more terminal-phosphate-labeled nucleotides with distinct labels.

63. The method of claim 62 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.

64. The method of claim 60 wherein said terminal-phosphate-labeled nucleotide may be represented by formula I:

65. The method of claim 64, wherein said terminal phosphate-labeled nucleotide comprises four or more phosphoryl groups in the phosphate chain.

66. The method of claim 64 wherein said phosphatase changes said phosphate ester linkage to produce said detectable species.

67. The method of claim 64 wherein said sugar moiety is selected from the group consisting of ribosyl, 2′

-deoxyribosyl, 3′

-deoxyribosyl, 2′

, 3′

-didehydrodideoxyribosyl, 2′

,3′

-dideoxyribosyl, 2′

-alkoxyribosyl, 2′

-azidoribosyl, 2′

-aminoribosyl, 2′

-fluororibosyl, 2′

mercaptoriboxyl, 2′

-alkylthioribosyl, carbocyclic, acyclic and other modified sugars.

68. The method of claim 64 wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, 7-deazaguanine, hypoxantinine, 7-deazahypoxanthine, adenine, 7-deazaadenine and 2,6-diaminopurine.

69. The method of claim 64 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.

70. The method of claim 60 wherein 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.

71. A method of detecting the presence of a nucleic acid sequence comprising the steps of:

(a) conducting a nucleic acid polymerase reaction in the presence of at least one terminal-phosphate-labeled nucleotide having four or more phosphate groups in the polyphosphate chain, which reaction results in the production of a labeled polyphosphate;

(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and

(c) detecting the presence of said detectable species.

72. The method of claim 71, wherein step (a) further comprises conducting said polymerase reaction in the presence of a phosphatase.

73. The method of claim 71, further comprising the step of quantifying said nucleic acid sequence.

74. The method of claim 71, wherein step (a) further comprises conducting said polymerase reaction in the presence of two or more terminal-phosphate-labeled nucleotides with distinct labels.