Detection of nucleic acids by type-specific hybrid capture method

• US 20040214302A1
• Filed: 04/04/2003
• Published: 10/28/2004
• Est. Priority Date: 06/15/2000
• Status: Active Grant

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Tang, Yanlin, Williams, Inna, Troy, John, Lorincz, Attila, Anthony, James
• Granted Patent: US 7,645,571 B2
• Time in Patent Office: Days
• US Class Current: 435/252.3
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/705 for herpetoviridae, e.g. herpes simplex, varicella zoster
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid target sequences. The method produces DNA/RNA hybrids which can be detected by a variety of methods.

Claims

1. A method of detecting a target nucleic acid comprising:

a) hybridizing a single-stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe to form double-stranded hybrids between said probes and the target nucleic acid, wherein the capture sequence probe and the signal sequence probe are capable of hybridizing to non-overlapping regions within the target nucleic acid and not hybridizing to each other;

b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes;

c) binding the hybrid to a solid phase to form a bound hybrid; and

d) detecting the bound hybrid.

3. The method of claim 1 or 2, wherein the signal sequence probe is single-stranded.

4. The method of claim 1 or 2, wherein the capture sequence probe is modified with at least one ligand.

5. The method of claim 4, wherein the ligand is biotin.

6. The method of claim 5, wherein the capture sequence probe is linear having a 5′

and 3′

end, wherein both the 5′ and

the 3′

ends are biotinylated.

7. The method of claim 1 or 2, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 3 kilobases apart.

8. The method of claim 1 or 2, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 500 bases apart.

9. The method of claim 1 or 2, wherein the capture sequence probe is a fusion of two or more sequences complementary to different regions of the target nucleic acid or to different target molecules.

10. The method of claim 1 or 2, wherein the double-stranded hybrid formed is a DNA/RNA hybrid.

11. The method of claim 1 or 2, further comprising the step of forming single-stranded or partially single-stranded target nucleic acid prior to the hybridization step.

12. The method of claim 1 or 2, wherein hybridization of the capture sequence probe and the signal sequence probe to the target nucleic acid are performed sequentially.

13. The method of claim 1, wherein step a) and step b) are performed simultaneously.

14. The method of claim 1, wherein step a) and step c) are performed simultaneously.

15. The method of claim 1, wherein steps a), b), and c) are performed simultaneously.

17. The method of claim 1 or 2, wherein the blocker probe has lower melting temperature than that of the capture sequence probe.

18. The method of claim 1, wherein the hybrid is captured onto a solid phase.

19. The method of claim 17, wherein the solid phase is coated with streptavidin.

20. The method of claim 17, wherein the solid phase is a microplate.

21. The method of claim 1 wherein step c) is carried out at room temperature.

22. The method of claim 1 or 2, wherein the bound hybrid is detected using an antibody that recognizes the hybrid.

23. The method of claim 22, wherein the hybrid is a DNA/RNA-hybrid.

24. The method of claim 22, wherein the antibody recognizes a DNA/RNA hybrid is labeled with alkaline-phosphatase.

54. The method according to claim 1, wherein the signal sequence probe comprises a first region having a DNA/RNA duplex structure and a second region having a single stranded nucleic acid sequence.

55. The method according to claim 54, wherein the DNA/RNA duplex is a M13 DNA-M13 RNA duplex.

2. A method of detecting a target nucleic acid comprising:

a) hybridizing a single-stranded or partially single-stranded target nucleic acid to an immobilized capture sequence probe and a signal sequence probe to form double-stranded hybrids between said probes and the target nucleic acid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other;

b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes;

c) detecting the bound hybrid.

16. The method of claim 2, wherein step a) and step b) are performed simultaneously.

25. A method of detecting a target nucleic acid comprising:

a) hybridizing a single-stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other, wherein said hybridization forms an RNA/DNA hybrid between said signal sequence probe and the target nucleic acid; and

b) detecting the RNA/DNA hybrid by binding an antibody that recognizes the RNA/DNA hybrid to said hybrid, wherein said antibody is detectably labeled.

26. The method of claim 25, further comprising binding the hybrid formed in step a) to a solid phase to form a bound hybrid.

27. The method of claim 25, wherein the signal sequence probe is single-stranded.

28. The method of claim 25, wherein the capture sequence probe is modified with at least one ligand.

29. The method of claim 28, wherein the capture sequence probe is biotinylated.

30. The method of claim 29, wherein the capture sequence probe is linear having a 5′

and a 3′

end, wherein both the 5′ and

the 3′

ends are biotinylated.

31. The method of claim 25, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 3 kilobases apart.

32. The method of claim 25, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 500 bases apart.

33. The method of claim 25, further comprising the step of forming single-stranded target nucleic acid prior to the hybridization step.

34. The method of claim 25, wherein hybridizations of the capture sequence probe and the signal sequence probe to the target nucleic acid are performed sequentially.

35. The method of claim 25, wherein the hybrid formed in step a) is bound onto a solid phase forming a bound hybrid.

36. The method of claim 35, wherein the bound hybrid is formed at room temperature.

37. The method of claim 35, wherein the solid phase is coated with streptavidin.

38. The method of claim 35, wherein the solid phase is a microplate.

39. The method of claim 25, wherein the antibody is labeled with alkaline-phosphatase.

40. The method of claim 25, further comprising adding a blocker probe to the hybridization step, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes.

41. The method of claim 40, wherein the blocker probes are added to the hybridization reaction following the hybridization of the capture sequence probes to the target nucleic acid.

42. The method of claim 40, wherein the blocker probe has a lower melting temperature than that of the capture sequence probe.

43. A method of detecting a target nucleic acid comprising:

a) hybridizing a single stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other, wherein the signal sequence probe comprises a DNA/RNA hybrid region, wherein said hybridization forms a complex; and

b) detecting said complex.

44. The method of claim 43 wherein the signal sequence probe comprises of at least 40 base pairs in length.

45. The method of claim 43 wherein the capture sequence probe comprises of at least 6 base pairs in length.

46. The method of claim 45 wherein the capture sequence probe is immobilized on a solid phase.

47. The method of claim 43 wherein said complex is detected by binding an antibody that recognizes the DNA/RNA hybrid region to said region, wherein the antibody is detectably labeled.

48. The method of claim 43 wherein the capture sequence is modified with at least one ligand.

49. The method of claim 48 wherein the ligand is biotin.

50. The method of claim 49, wherein the capture sequence probe is linear having a 5′

and 3′

end, wherein both the 5′ and

3′

ends are biotinylated.

51. The method of claim 43, further comprising adding a blocker probe after the hybridization step, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probe.

52. The method of claim 51, wherein the blocker probes comprise a length of 4-10 base pairs shorter than the length of the capture sequence probe.

53. A nucleic acid probe consisting of a sequence selected from the group consisting of SEQ ID NO:

1 through SEQ ID NO;

160.

56. A method of detecting a target nucleic acid comprising:

a) hybridizing a single-stranded or partially single-stranded target nucleic acid to a capture sequence probe, a bridge probe and a signal sequence probe to form double-stranded hybrids , wherein the capture sequence probe and the bridge probe hybridizes to non-overlapping regions within the target nucleic acid and not to each other, and the signal sequence probe hybridizes to the bridge probe and not to the target nucleic acid and the capture sequence probe;

b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes;

c) binding the hybrid to a solid phase to form a bound hybrid; and

d) detecting the bound hybrid.

57. The method according to claim 56, wherein the signal sequence probe comprises a first region having a DNA/RNA duplex structure and a second region having a single stranded nucleic acid.

58. The method according to claim 57, wherein the DNA/RNA duplex is a M13 DNA-M13 RNA duplex.

59. The method according to claim 57, wherein the DNA/RNA duplex is a hybrid formed between repeat sequences within the signal sequence probe and a nucleic acid molecule having complementary sequences to the repeat sequences.

60. The method according to claim 56, wherein the bridge probe further comprises a poly(A) tail.

61. The method according to claim 60, wherein the signal sequence probe comprises a single stranded poly(dT) DNA sequence which hybridizes to the poly(A) tail of the bridge probe, and a DNA/RNA duplex fonned between the poly(dT) sequences in the signal sequence probe and a nucleic acid molecule having poly(A) sequences.

62. A method of detecting a target nucleic acid comprising:

a) hybridizing a single-stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe to form double-stranded hybrids between said probes and the target nucleic acid;

b) binding the hybrid to a solid phase forming a bound hybrid; and

c) detecting the bound hybrid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other.

63. The method of claim 62, wherein hybridization of the capture sequence probe and the signal sequence probe to the target nucleic acid are performed sequentially.

64. The method of claim 62, wherein step a) and step b) are performed simultaneously.

65. The method of claim 62, further comprising adding a blocker probe, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes.

66. The method of claim 65, wherein the addition of the blocker probe and hybridization step are performed simultaneously.

67. The method of claim 65, wherein the addition of the blocker probe and binding step are performed simultaneously.

68. The method of claim 62, wherein the signal sequence probe is single-stranded.

69. The method of claim 62, wherein the capture sequence probe is modified with at least one ligand.

70. The method of claim 62, wherein the ligand is biotin.

71. The method of claim 70, wherein the capture sequence probe is linear having a 5′

and 3′

end, wherein both the 5′ and

the 3′

ends are biotinylated.

72. The method of claim 62, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 3 kilobases apart.

73. The method of claim 62, wherein the capture sequence probe and the signal sequence probe hybridize to regions of the target nucleic acid, wherein the regions are less than 500 bases apart.

74. The method of claim 62, wherein the capture sequence probe is a fusion of two or more sequences complementary to different regions of the target nucleic acid or to different target molecules.

75. The method of claim 62, wherein the double-stranded hybrid formed is a DNA/RNA hybrid.

76. The method of claim 62, frher comprising the step of forming single-stranded DNA prior to the hybridization step.

77. The method of claim 62, wherein hybridization of the capture sequence probe and the signal sequence probe to the target nucleic acid are performed sequentially.

78. The method of claim 62, wherein step a) and step b) are performed simultaneously.

79. The method of claim 63, wherein the blocker probe has lower melting temperature than that of the capture sequence probe.

80. The method of claim 63, wherein the solid phase is coated with streptavidin.

81. The method of claim 73, wherein the solid phase is a microplate.

82. The method of claim 62, wherein step b) is carried out at about 20°

C. to 90°

C.

83. The method of claim 82, wherein the capture sequence probe comprises at least 6 bases in length.

84. The method of claim 62, wherein step b) is carried out at room temperature.

85. The method of claim 62, wherein the bound hybrid is detected using an antibody that recognizes a hybrid.

86. The method of claim 85, wherein the hybrid is a DNA/RNA-hybrid.

87. The method of claim 85, wherein the antibody that recognizes a DNA/RNA hybrid is labeled with alkaline-phosphatase.

88. The method according to claim 62, wherein the signal sequence probe comprises a deleted capture sequence probe region and is complementary to the target nucleic acid.

89. The method according to claim 62, wherein the target nucleic acid is a single nucleotide polymorphism.

90. The method according to claim 89, wherein the specificity of binding the hybrid to a solid phase is modulated by temperatures of greater than room temperature.

91. The method according to claim 89, wherein the specificity of binding the hybrid to a solid phase is modulated by the addition of blocker probes.

92. The method according to claim 89, wherein the specificity of binding the hybrid to a solid phase is modulated by temperatures of greater than room temperature and the addition of blocker probes.