Electrochemical detection of nucleic acid hybridization

• US 6,361,951 B1
• Filed: 06/26/2000
• Issued: 03/26/2002
• Est. Priority Date: 06/27/1995
• Status: Expired due to Term

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Johnston, Dean H., Thorp, H. Holden, Napier, Mary E., Loomis, Carson R., Sistare, Mark F., Kim, Jinheung
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/603,217
• Time in Patent Office: 638 Days
• Field of Search: 435/6, 435/91.2, 536/27, 536/28, 536/29, 546/2
• US Class Current: 435/6.13
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method of detecting a nucleic acid (e.g., DNA, RNA) that contains at least one preselected base (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine) comprises (a) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; and (c) determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction at the preselected base. The method may be used in a variety of applications, including DNA sequencing, diagnostic assays, and quantitative analysis.

Claims

1. A method of detecting DNA hybridization comprising:

(a) contacting a DNA sample with an oligonucleotide probe to form a hybridized DNA;

(b) reacting said hybridized DNA with a transition metal complex that oxidizes a preselected base in said oligonucleotide probe in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and the preselected base, said oligonucleotide probe having at least one of said preselected bases, from which preselected base there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(c) detecting said oxidation-reduction reaction;

(d) determining the presence or absence of hybridized DNA from said detected oxidation-reduction reaction at said preselected base.

2. The method according to claim 1, wherein said determining step further comprises the steps of:

(i) measuring the reaction rate of said detected oxidation-reduction reaction, (ii) comparing said measured reaction rate to the oxidation-reduction reaction rate of the transition metal complex with a single-stranded DNA; and

then (iii) determining whether said measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded DNA.

3. The method according to claim 1, wherein said DNA sample is a single-stranded DNA sample, and said hybridized DNA is a duplex.

4. The method according to claim 1, wherein said oligonucleotide probe includes from about 4 to about 100 bases.

5. The method according to claim 1, wherein said preselected base is guanine.

6. The method according to claim 1, wherein said preselected base is adenine.

7. The method according to claim 1, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃²⁺, Ru(Me₂-bpy)₃²⁺, Ru(Me₂-phen)₃²⁺, Fe(bpy)₃²⁺, Fe(5-Cl-phen)₃²⁺, Os(bpy)₃²⁺, Os(5-Cl-phen)₃²⁺, and ReO₂(py)₄¹⁺.

8. The method according to claim 1, wherein said reacting step comprises reacting said transition metal complex with said hybridized DNA sample under conditions sufficient to effect the selective oxidation of said preselected base.

9. The method according to claim 1, further comprising the step of amplifying said hybridized DNA prior to said contacting step.

10. The method according to claim 9, wherein said step of amplifying said DNA sample is carried out by polymerase chain reaction, strand displacement amplification, ligase chain reaction, or nucleic acid sequence-based amplification.

11. The method according to claim 2, wherein said step of measuring the reaction rate of said oxidation-reduction reaction comprises measuring the cyclic voltammogram of the reaction.

12. The method according to claim 2, wherein said step of comparing comprises comparing the cyclic voltammogram of the reaction of the transition metal complex with the hybridized DNA sample against the known cyclic voltammogram of the reaction of the transition metal complex with single-stranded DNA.

13. The method according to claim 1, wherein said oligonucleotide probe is immobilized on a solid surface.

14. The method according to claim 13, wherein said transition metal complex is immobilized on said solid surface.

83. The method according to claim 13 wherein said solid surface comprises an electrode.

15. A method of sequencing DNA comprising

(a) contacting a DNA sample with an oligonucleotide probe to form a hybridized DNA, said oligonucleotide probe including a preselected base having a unique oxidation rate;

b) reacting said hybridized DNA with a transition metal complex that oxidizes said preselected base in said oligonucleotide probe in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and the preselected base, said oligonucleotide probe having a predetermined number of said preselected base, from which preselected base there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(c) detecting said oxidation-reduction reaction;

(d) measuring the reaction rate of said detected oxidation-reduction reaction; and

(e) identifying the base paired with said preselected base.

16. The method according to claim 15, wherein said identifying step comprises (i) comparing said measured reaction rate to each of the four different known oxidation-reduction reaction rates of the transition metal complex with a DNA having adenine, cytosine, guanine, or thymine bound to said preselected base;

and (ii) determining which of said known oxidation-reduction reaction rates is essentially the same as said measured reaction rate.

17. The method according to claim 15, wherein said oligonucleotide probe further includes a second preselected base having a unique oxidation rate, wherein the oxidation rate of said second preselected base is different from the oxidation rate of said preselected base.

18. The method according to claim 17, wherein said detecting step further comprises detecting the oxidation-reduction reaction of the transition metal complex with said second preselected base;

wherein said measuring step further comprises measuring the reaction rate of said detected oxidation-reduction reaction of the transition metal complex with said second preselected base; and

wherein said identifying step further comprises identifying the base paired with said second preselected base.

19. The method according to claim 15, further comprising repeating steps (a) through (e) with a sufficient number of oligonucleotide probes having said preselected base at different sites to identify each base in said DNA sample.

84. The method according to claim 15 wherein said detection of said oxidation/reduction reaction in step (c) comprises the use of an electrode.

20. A method of detecting RNA hybridization comprising:

(a) contacting an RNA sample with an oligonucleotide probe to form a hybridized RNA;

(b) reacting said hybridized RNA with a transition metal complex that oxidizes a preselected base in said oligonucleotide probe in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and the preselected base, said oligonucleotide probe having at least one of said preselected base, from which preselected base there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(c) detecting said oxidation-reduction reaction;

(d) determining the presence or absence of hybridized RNA from said detected oxidation-reduction reaction at said preselected base.

21. The method according to claim 20, wherein said determining step further comprises the steps of:

(i) measuring the reaction rate of said detected oxidation-reduction reaction, (ii) comparing said measured reaction rate to the oxidation-reduction reaction rate of the transition metal complex with a single-stranded RNA; and

then (iii) determining whether said measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded RNA.

22. The method according to claim 20, wherein said RNA sample is a single-stranded RNA sample, and said hybridized RNA is a duplex.

23. The method according to claim 20, wherein said oligonucleotide probe includes from about 4 to about 100 bases.

24. The method according to claim 20, wherein said preselected base is guanine.

25. The method according to claim 20, wherein said preselected base is adenine.

26. The method according to claim 20, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃²⁺, Ru(Me₂-bpy)₃²⁺, Ru(Me₂-phen)₃²⁺, Fe(bpy)₃²⁺, Fe(5-Cl-phen)₃²⁺, Os(bpy)₃²⁺, Os(5-Cl-phen)₃²⁺, and ReO₂(py)₄¹⁺.

27. The method according to claim 20, wherein said reacting step comprises reacting said transition metal complex with said hybridized RNA sample under conditions sufficient to effect the selective oxidation of said preselected base.

28. The method according to claim 20, further comprising the step of amplifying said hybridized RNA prior to said contacting step.

29. The method according to claim 28, wherein said step of amplifying said RNA sample is carried out by reverse-transcriptase polymerase chain reaction.

30. The method according to claim 21, wherein said step of measuring the reaction rate of said oxidation-reduction reaction comprises measuring the cyclic voltammogram of the reaction.

31. The method according to claim 21, wherein said step of comparing comprises comparing the cyclic voltammogram of the reaction of the transition metal complex with the hybridized RNA sample against the known cyclic voltammogram of the reaction of the transition metal complex with single-stranded RNA.

32. The method according to claim 20, wherein said oligonucleotide probe is immobilized on a solid surface.

33. The method according to claim 32, wherein said transition metal complex is immobilized on said solid surface.

85. The method according to claim 32 wherein the solid surface comprises an electrode.

34. A method of sequencing RNA comprising:

(a) contacting an RNA sample with an oligonucleotide probe to form a hybridized RNA, said oligonucleotide probe including a preselected base having a unique oxidation rate;

(b) reacting said hybridized RNA with a transition metal complex that oxidizes said preselected base in said oligonucleotide probe in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and the preselected base, said oligonucleotide probe having a predetermined number of said preselected base, from which preselected base there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(c) detecting said oxidation-reduction reaction;

(d) measuring the reaction rate of said detected oxidation-reduction reaction; and

(e) identifying the base paired with said preselected base.

35. The method according to claim 34, wherein said identifying step comprises (i) comparing said measured reaction rate to each of the four different known oxidation-reduction reaction rates of the transition metal complex with an RNA having adenine, cytosine, guanine, or uracil bound to said preselected base;

and (ii) determining which of said known oxidation-reduction reaction rates is essentially the same as said measured reaction rate.

36. The method according to claim 34, wherein said oligonucleotide probe further includes a second preselected base having a unique oxidation rate, wherein the oxidation rate of said second preselected base is different from the oxidation rate of said preselected base.

37. The method according to claim 36, wherein said detecting step further comprises detecting the oxidation-reduction reaction of the transition metal complex with said second preselected base;

wherein said measuring step further comprises measuring the reaction rate of said detected oxidation-reduction reaction of the transition metal complex with said second preselected base; and

wherein said identifying step further comprises identifying the base paired with said second preselected base.

38. The method according to claim 34, further comprising repeating steps (a) through (e) with a sufficient number of oligonucleotide probes having said preselected base at different sites to identify each base in said RNA sample.

86. The method according to claim 34 wherein said detection of said oxidation/reduction reaction in step (c) comprises the use of an electrode.

39. A method of detecting a nucleic acid, said nucleic acid containing at least one preselected base, said method comprising:

(a) reacting said nucleic acid with a transition metal complex that oxidizes said preselected base in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and the preselected base, from which preselected base there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(b) detecting said oxidation-reduction reaction; and

(c) determining the presence or absence of said nucleic acid from said detected oxidation-reduction reaction at said preselected base.

40. A method according to claim 39, wherein said reacting step is preceded by the step of:

contacting said nucleic acid with a complementary nucleic acid to form a hybridized nucleic acid.

41. The method according to claim 40, wherein said determining step further comprises the steps of:

(i) measuring the reaction rate of said detected oxidation-reduction reaction, (ii) comparing said measured reaction rate to the oxidation-reduction reaction rate of the transition metal complex with a single-stranded nucleic acid; and

then (iii) determining whether said measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded nucleic acid.

42. The method according to claim 41, wherein said step of measuring the reaction rate of said oxidation-reduction reaction comprises measuring the cyclic voltammogram of the reaction.

43. The method according to claim 41, wherein said step of comparing comprises comparing the cyclic voltammogram of the reaction of the transition metal complex with the hybridized nucleic acid sample against the known cyclic voltammogram of the reaction of the transition metal complex with single-stranded nucleic acid.

44. The method according to claim 39, wherein said nucleic acid includes from about 4 to about 100 bases.

45. The method according to claim 39, wherein said preselected base is selected from the group consisting of guanine and adenine.

46. The method according to claim 39, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃²⁺, Ru(Me₂-bpy)₃²⁺, Ru(Me₂-phen)₃²⁺, Fe(bpy)₃²⁺, Fe(5-Cl-phen)₃²⁺, Os(bpy)₃²⁺, Os(5-Cl-phen)₃²⁺, and ReO₂(py)₄¹⁺.

47. A method according to claim 39, wherein said nucleic acid is DNA.

48. A method according to claim 39, wherein said nucleic acid is RNA.

49. The method according to claim 39, further comprising the step of amplifying said nucleic acid prior to said reacting step.

50. The method according to claim 49, wherein said step of amplifying said nucleic acid is carried out by polymerase chain reaction, strand displacement amplification, ligase chain reaction, or nucleic acid sequence-based amplification.

51. The method according to claim 39, wherein said nucleic acid is immobilized on a solid surface.

52. The method according to claim 51, wherein said transition metal complex is immobilized on said solid surface.

69. The method according to claim 40, wherein the preselected base is selected from the group consisting of guanine, adenine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine.

70. A method according to claim 69, wherein said reacting step is preceded by the step of:

contacting said nucleic acid with a complementary nucleic acid to form a hybridized nucleic acid.

73. The method according to claim 69, wherein said determining step is a quantitatively determining step.

74. The method according to claim 69, wherein said nucleic acid is DNA.

75. The method according to claim 69, wherein said nucleic acid is RNA.

76. The method according to claim 69, wherein said preselected base is selected from the group consisting of guanine and adenine.

77. The method according to claim 69, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃²⁺, Ru(Me₂-bpy)₃²⁺, Ru(Me₂-phen)₃²⁺, Fe(bpy)₃²⁺, Fe(5-Cl-phen)₃²⁺, Os(bpy)₃²⁺, Os(5-Cl-phen)₃²⁺, and ReO₂(py)₄¹⁺.

87. The method according to claim 40 wherein said complementary nucleic acid is immobilized on a solid surface.

88. The method according to claim 87 wherein said solid surface comprises an electrode.

53. A method of detecting the presence or absence of a target nucleic acid in a test sample suspected of containing the same, wherein said target nucleic acid contains at least one preselected base, said method comprising:

(a) contacting said test sample to an oligonucleotide probe that specifically binds to said target nucleic acid to form a hybridized nucleic acid;

(b) contacting said hybridized nucleic acid to a transition metal complex that oxidizes said preselected base in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and the preselected base, from which preselected base there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(c) detecting the presence or absence of said oxidation-reduction reaction associated with said hybridized nucleic acid; and

(d) determining the presence or absence of said target nucleic acid in said test sample from said detected oxidation-reduction reaction at said preselected base.

54. A method according to claim 53, further comprising the step of:

separating said test sample from said hybridized nucleic acid prior to said detecting step.

55. A method according to claim 53, wherein said target nucleic acid is longer than said oligonucleotide probe, and wherein at least one of said preselected base is not hybridized to said oligonucleotide probe in said hybridized nucleic acid.

56. A method according to claim 54, wherein said determining step is a quantiatively determining step.

57. The method according to claim 54, wherein said determining step further comprises the steps of:

(i) measuring the reaction rate of said detected oxidation-reduction reaction, (ii) comparing said measured reaction rate to the oxidation-reduction reaction rate of the transition metal complex with a single-stranded target nucleic acid; and

then (iii) determining whether said measured reaction rate is essentially the same as the oxidation-reduction reaction rate of the transition metal complex with single-stranded target nucleic acid.

58. The method according to claim 57, wherein said step of measuring the reaction rate of said oxidation-reduction reaction comprises measuring the cyclic voltammogram of the reaction.

59. The method according to claim 57, wherein said step of comparing comprises comparing the cyclic voltammogram of the reaction of the transition metal complex with the hybridized target nucleic acid sample against the known cyclic voltammogram of the reaction of the transition metal complex with single-stranded target nucleic acid.

60. The method according to claim 53, wherein said target nucleic acid includes from about 4 to about 100 bases.

61. The method according to claim 53, wherein said preselected base is selected from the group consisting of guanine and adenine.

62. The method according to claim 53, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃²⁺, Ru(Me₂-bpy)₃²⁺, Ru(Me₂-phen)₃²⁺, Fe(bpy)₃²⁺, Fe(5-Cl-phen)₃²⁺, Os(bpy)₃²⁺, Os(5-Cl-phen)₃²⁺, and ReO₂(py)₄¹⁺.

63. A method according to claim 53, wherein said target nucleic acid is DNA.

64. A method according to claim 53, wherein said target nucleic acid is RNA.

65. The method according to claim 53, further comprising the step of amplifying said target nucleic acid prior to said reacting step.

66. The method according to claim 65, wherein said step of amplifying said target nucleic acid sample is carried out by polymerase chain reaction, strand displacement amplification, ligase chain reaction, or nucleic acid sequence-based amplification.

67. The method according to claim 53, wherein said oligonucleotide probe is immobilized on a solid surface.

68. The method according to claim 67, wherein said transition metal complex is immobilized on said solid surface.

89. The method according to claim 67 wherein said solid surface comprises an electrode.

71. A method of detecting a nucleic acid, said nucleic acid containing at least one preselected base, said method comprising:

(a) contacting a sample containing said nucleic acid to an electrode, said electrode comprising (i) a conductive substrate having a working surface formed thereon; and

(ii) a nonconductive polymer layer connected to said working surface, wherein said polymer layer binds said nucleic acid thereto;

(b) reacting said nucleic acid with a transition metal complex that oxidizes said preselected base in an oxidation-reduction reaction under conditions that cause an oxidation-reduction reaction between the transition metal complex and the preselected base, from which preselected base there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle, and wherein said polymer layer is porous to said transition metal complex;

(c) detecting said oxidation-reduction reaction by measuring current flow through said electrode; and

(d) determining the presence or absence of said nucleic acid from said detected oxidation-reduction reaction at said preselected base.

72. A method according to claim 71, wherein said reacting step is preceded by the step of:

contacting said nucleic acid with a complementary nucleic acid to form a hybridized nucleic acid.

78. The method according to claim 71, wherein said determining step is a quantitatively determining step.

79. The method according to claim 71, wherein said nucleic acid is DNA.

80. The method according to claim 71, wherein said nucleic acid is RNA.

81. The method according to claim 71, wherein said preselected base is selected from the group consisting of guanine and adenine.

82. The method according to claim 71, wherein said transition metal complex is selected from the group consisting of Ru(bpy)₃²⁺, Ru(Me₂-bpy)₃²⁺, Ru(Me₂-phen)₃²⁺, Fe(bpy)₃²⁺, Fe(5-Cl-phen)₃²⁺, Os(bpy)₃²⁺, Os(5-Cl-phen)₃²⁺, and ReO₂(py)₄¹⁺.

90. The method according to claim 72 wherein the complementary nucleic acid is immobilized on said polymer layer.