Reactions on a solid surface

• US 20050164177A1
• Filed: 12/08/2000
• Published: 07/28/2005
• Est. Priority Date: 11/29/1996
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Neri, Bruce P., Hall, Jeff G., Smith, Lloyd M., Lyamichev, Victor
• Granted Patent: US 7,432,048 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present invention relates to compositions and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for forming a nucleic acid cleavage structure on a solid support and cleaving the nucleic acid cleavage structure in a site-specific manner. For example, in some embodiments, a 5′ nuclease activity from any of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

Claims

1. A system comprising oligonucleotides capable of hybridizing to a target nucleic acid to form an invasive cleavage structure and a solid support;

wherein one or more of said oligonucleotides is attached to said solid support.

2. The system of claim 1, further comprising an agent for detecting the presence of said invasive cleavage structure.

3. The system of claim 2, wherein said agent comprises a cleavage agent.

4. The system of claim 2, wherein said cleavage agent comprises a structure-specific nuclease.

5. The system of claim 4, wherein said agent comprises a 5′

nuclease.

6. The system of claim 5, wherein said 5′

nuclease comprises a FEN-1 endonuclease.

7. The system of claim 5, wherein said 5′

nuclease comprises a polymerase.

8. The system of claim 1, wherein said oligonucleotides comprise first and second oligonucleotides, said first oligonucleotide comprising a portion complementary to a first region of said target nucleic acid and said second oligonucleotide comprising a 3′

portion and a 5′

portion, said 5′

portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid.

9. The system of claim 8, wherein one or more of said first oligonucleotides is attached to said solid support.

10. The system of claim 8, wherein one or more of said second oligonucleotides is attached to said solid support.

11. The system of claim 8, wherein one or more of said first oligonucleotides and one or more of said second oligonucleotides is attached to said solid support.

12. The system of claim 8, wherein said first oligonucleotides comprise signal probe oligonucleotides.

13. The system of claim 8, wherein said first oligonucleotides comprise a fluorescent dye.

14. The system of claim 13, wherein said first oligonucleotides further comprise a quencher molecule.

15. The system of claim 1, further comprising a spacer molecule, wherein said one or more oligonucleotides is attached to said solid support through said spacer molecule.

16. The system of claim 15, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

17. The system of claim 1, wherein said solid support comprises a glass solid support.

18. The system of claim 1, wherein said solid support comprises a latex solid support.

19. The system of claim 1, wherein said solid support comprises a hydrogel solid support.

20. The system of claim 1, wherein said target nucleic acid is attached to said solid support.

21. The system of claim 2, wherein said agent is attached to said solid support.

22. The system of claim 1, wherein said solid support comprises a bead.

23. The system of claim 1, wherein said solid support comprises a multi-well plate.

24. The system of claim 1, wherein said solid support comprises a column.

25. The system of claim 1, wherein said solid support comprises a microarray.

26. The system of claim 1, wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.

42. The method of claim 1, further providing a spacer molecule, wherein said one or more oligonucleotides is attached to said solid support through said spacer molecule.

43. The method of claim 42, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

27. A method for characterizing a nucleic acid comprising:

a) providing;

i. a sample suspected of containing a target nucleic acid;

ii. oligonucleotides capable of hybridizing to said target nucleic acid to form an invasive cleavage structure;

iii. a solid support, wherein one or more of said oligonucleotides is attached to said solid support; and

iv. an agent capable of detecting the presence of an invasive cleavage structure; and

b) exposing said sample to said oligonucleotides and said agent.

28. The method of claim 27, wherein said exposing said sample to said oligonucleotides and said agent comprises exposing said sample to said oligonucleotides and said agent under conditions wherein an invasive cleavage structure is formed between a target nucleic acid and said oligonucleotides if said target nucleic acid is present in said sample.

29. The method of claim 28, further comprising the step of c) detecting said invasive cleavage structure.

30. The method of claim 27, wherein said agent comprises a cleavage agent.

31. The method of claim 30, wherein said cleavage agent comprises a structure-specific nuclease.

32. The method of claim 30, wherein said agent comprises a 5′

nuclease.

33. The method of claim 32, wherein said 5′

nuclease comprises a FEN-1 endonuclease.

34. The method of claim 32, wherein said 5′

nuclease comprises a polymerase.

35. The method of claim 27, wherein said oligonucleotides comprise first and second oligonucleotides, said first oligonucleotide comprising a portion complementary to a first region of said target nucleic acid and said second oligonucleotide comprising a 3′

portion and a 5′

portion, said 5′

portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid.

36. The method of claim 35, wherein one or more of said first oligonucleotides is attached to said solid support.

37. The method of claim 35, wherein one or more of said second oligonucleotides is attached to said solid support.

38. The method of claim 35, wherein one or more of said first oligonucleotides and one or more of said second oligonucleotides is attached to said solid support.

39. The method of claim 35, wherein said first oligonucleotides comprise signal probe oligonucleotides.

40. The method of claim 35, wherein said first oligonucleotides comprise a fluorescent dye.

41. The method of claim 40, wherein said first oligonucleotides further comprise a quencher molecule.

44. The method of claim 27, wherein said solid support comprises a glass solid support.

45. The method of claim 27, wherein said solid support comprises a latex solid support.

46. The method of claim 27, wherein said solid support comprises a hydrogel solid support.

47. The method of claim 27, wherein said target nucleic acid is attached to said solid support.

48. The method of claim 27, wherein said agent is attached to said solid support.

49. The method of claim 27, wherein said solid support comprises a bead.

50. The method of claim 27, wherein said solid support comprises a multi-well plate.

51. The method of claim 27, wherein said solid support comprises a column.

52. The method of claim 27, wherein said solid support comprises a microarray.

53. The method of claim 27, wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.

54. A method for cleaving multiple oligonucleotides comprising:

a) providing;

i. a plurality of first oligonucleotides attached to a solid support;

ii. a second oligonucleotide attached to said solid support; and

iii. a cleavage agent; and

b) exposing said solid support to said cleavage agent under conditions such that;

i. a first cleavage structure is formed, said first cleavage structure comprising one of said first oligonucleotides and said second oligonucleotide;

ii. said cleavage agent cleaves said first oligonucleotide in said first cleavage structure to produce a first cleavage fragment;

iii. after cleavage of said first first oligonucleotide, a second cleavage structure is formed, said second cleavage structure comprising a second of said first oligonucleotides and said second oligonucleotide;

iv. said cleavage agent cleaves said first oligonucleotide in said second cleavage structure to produce a second cleavage fragment.

55. The method of claim 54, wherein said conditions comprise exposing a target nucleic acid to said solid support.

56. The method of claim 55, wherein said first oligonucleotides comprise a portion complementary to a first region of said target nucleic acid;

and wherein said second oligonucleotide comprises a 3′

portion and a 5′

portion, said 5′

portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid.

57. The method of claim 54, further comprising the step of c) detecting the presence of said first or said second cleavage fragments.

58. The method of claim 54, wherein said cleavage agent comprises a structure-specific nuclease.

59. The method of claim 54, wherein said cleavage agent comprises a 5′

nuclease.

60. The method of claim 59, wherein said 5′

nuclease comprises a FEN-1 endonuclease.

61. The method of claim 59, wherein said 5′

nuclease comprises a polymerase.

62. The method of claim 54, wherein said second oligonucleotide is attached to said solid support through a spacer molecule.

63. The method of claim 62, wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with three or more of said first oligonucleotides.

64. The method of claim 63, wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with ten or more of said first oligonucleotides.

65. The method of claim 62, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

66. The method of claim 54, wherein said solid support comprises a glass solid support.

67. The method of claim 54, wherein said solid support comprises a latex solid support.

68. The method of claim 54, wherein said solid support comprises a hydrogel solid support.

69. The method of claim 54, wherein said solid support comprises a bead.

70. The method of claim 54, wherein said solid support comprises a multi-well plate.

71. The method of claim 54, wherein said solid support comprises a column.

72. The method of claim 54, wherein said solid support comprises a micro array.

73. The method of claim 54, wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.

74. A composition comprising a solid support;

said solid support attached to a a plurality of first oligonucleotides and a second oligonucleotide, wherein said second oligonucleotide is capable of forming an invasive cleavage structure with each of said plurality of first oligonucleotides in the presence of a target nucleic acid.

75. The composition of claim 75, wherein said plurality of first oligonucleotides comprises three or more first oligonucleotides.

76. The composition of claim 75, wherein said plurality of first oligonucleotides comprises ten or more first oligonucleotides.

77. The composition of claim 75, wherein said first oligonucleotides comprise a portion complementary to a first region of said target nucleic acid;

and wherein said second oligonucleotide comprises a ₃′

portion and a 5′

portion, said 5′

portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid.

78. The composition of claim 75, wherein said solid support is further attached to a cleavage agent.

79. The composition of claim 75, wherein said solid support is further attached to said target nucleic acid.

80. The composition of claim 75, wherein said second oligonucleotide is attached to said solid support through a spacer molecule.

82. The composition of claim 75, wherein said solid support comprises a glass solid support.

83. The composition of claim 75, wherein said solid support comprises a latex solid support.

84. The composition of claim 75, wherein said solid support comprises a hydrogel solid support.

85. The composition of claim 75, wherein said solid support comprises a bead.

86. The composition of claim 75, wherein said solid support comprises a multi-well plate.

87. The composition of claim 75, wherein said solid support comprises a column.

88. The composition of claim 75, wherein said solid support comprises a microarray.

89. The composition of claim 75, wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.

81. The composition of claim 81, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.