Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof

• US 7,771,934 B2
• Filed: 05/28/2004
• Issued: 08/10/2010
• Est. Priority Date: 12/13/2000
• Status: Expired due to Fees

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US10/857,160
• Publication Number:
  US 20050064456A1
• Time in Patent Office: 2,265 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension

Abstract

The present invention provides novel isothermal methods of generating multiple copies of, detecting and/or quantifying nucleic acid sequences of interest based on limited primer extension or attachment of oligonucleotide pairs using composite RNA/DNA primers. Methods for generating multiple copies of and/or detecting and/or quantifying nucleic acid sequences, wherein products of primer extension or attachment of oligonucleotide pairs comprising a cleavable portion are generated, and wherein cleavage of the products results in dissociation of cleaved products from target polynucleotides, are provided. The invention further provides compositions, kits and systems for practicing these methods.

Claims

1. A method of linearly generating multiple copies of a nucleic acid sequence of interest from a template polynucleotide, said method comprising the steps of:

(a) hybridizing a first oligonucleotide and a second oligonucleotide to non-overlapping portions of the target polynucleotide, wherein a portion of the target polynucleotide that is hybridizable to the first oligonucleotide is 3′

with respect to the portion of the target nucleotide that is hybridizable to the second oligonucleotide, wherein at least one of said oligonucleotides is a composite primer comprising an RNA portion and a DNA portion such that (i) if said first oligonucleotide is the composite primer, said RNA portion is at the 5′

end of said first oligonucleotide, and (ii) if said second oligonucleotide is the composite primer, said RNA portion is at the 3′

end of said second oligonucleotide, and wherein at least one of said first and second oligonucleotides comprises a sequence that is hybridizable to at least one nucleotide of the sequence of interest;

(b) optionally extending the first oligonucleotide with a DNA polymerase that lacks strand displacement activity;

(c) attaching the first oligonucleotide and second oligonucleotide to each other when hybridized to said target polynucleotide to generate an attached oligonucleotide combination product; and

(d) cleaving the RNA portion of the attached oligonucleotide combination product of (c) with an enzyme that cleaves RNA from an RNA/DNA hybrid such that the cleaved oligonucleotide combination product dissociates from the target polynucleotide,

wherein the attached oligonucleotide combination product is of a size that when the RNA is cleaved from the attached oligonucleotide combination product, the cleaved attached oligonucleotide product dissociates from the target polynucleotide, without strand displacement by an enzyme, under essentially the same conditions as those for hybridization of the composite oligonucleotides to the target and the attachment of the hybridized oligonucleotides,

(e) repeating steps (a)-(d) whereby multiple copies of the sequence of interest are produced, wherein said first or second oligonucleotide hybridize only to the template polynucleotide and not to said cleaved oligonucleotide combination product.

2. The method of claim 1, wherein the first oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

3. The method of claim 1, wherein the 3′

most nucleotide of the first oligonucleotide is hybridizable to the sequence of interest.

4. The method of claim 1, wherein the second oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

5. The method of claim 1, wherein the 5′

most nucleotide of the second oligonucleotide is hybridizable to at least one nucleotide of the sequence of interest.

6. The method of claim 1, wherein the sequence of interest is a single nucleotide base.

7. The method of claim 1, wherein the sequence of interest is more than a single nucleotide base.

8. The method of claim 1, wherein the first and second oligonucleotide each comprise a sequence hybridizable to at least one nucleotide of the sequence of interest.

9. The method of claim 1, wherein the enzyme that cleaves RNA is RNase H.

10. The method of claim 1, wherein the target polynucleotide is DNA or cDNA.

11. The method of claim 1, wherein the target polynucleotide is amplified.

12. The method of claim 11, wherein amplification occurs simultaneously with generating multiple copies of a sequence of interest.

13. The method of claim 11, wherein amplification occurs prior to generating multiple copies of a sequence of interest.

14. The method of claim 1, wherein the attaching is covalent.

15. The method of claim 14, wherein the covalent attachment is effected by ligase.

86. The method of claim 1, wherein the cleaved attached oligonucleotide product is between 2 and 24 continuous nucleotides in length.

16. A method of determining whether a nucleic acid sequence of interest is present or absent in a template polynucleotide, said method comprising the steps of:

(a) hybridizing a first oligonucleotide and a second oligonucleotide to non-overlapping portions of the target polynucleotide, wherein the portion of the target polynucleotide that is hybridizable to the first oligonucleotide is 3′

with respect to the portion of the target nucleotide that is hybridizable to the second oligonucleotide, wherein at least one of said oligonucleotides is a composite primer comprising an RNA portion and a DNA portion such that (i) if said first oligonucleotide is the composite primer, said RNA portion is at the 5′

end of said first oligonucleotide, and (ii) if said second oligonucleotide is the composite primer, said RNA portion is at the 3′

end of said second oligonucleotide, and wherein at least one of said oligonucleotides comprises a sequence that is hybridizable to at least one nucleotide of the sequence of interest;

(b) optionally extending the first oligonucleotide with a DNA polymerase that lacks strand displacement-activity;

(c) attaching the first oligonucleotide and second oligonucleotide to each other when hybridized to said target polynucleotide to generate an attached oligonucleotide combination product comprising a detectable identifying characteristic, whereby the attached oligonucleotide combination product is produced if the sequence of interest is present; and

(d) cleaving the RNA portion of the attached oligonucleotide combination product of (c), if any, with an enzyme that cleaves RNA from an RNA/DNA hybrid such that the cleaved attached oligonucleotide combination product dissociates from the target polynucleotide,

wherein the attached oligonucleotide combination product is of a size that when the RNA is cleaved the cleaved attached oligonucleotide combination product dissociates from the target polynucleotide, without strand displacement by an enzyme, under essentially the same conditions as those for attachment of the oligonucleotides, whereby detection of the cleaved attached oligonucleotide combination product comprising the detectable identifying characteristic indicates the presence of the sequence of interest, and wherein said first or second oligonucleotide hybridize only to the template polynucleotide and not to said cleaved oligonucleotide combination product.

17. The method of claim 16, wherein the first oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

18. The method of claim 16, wherein the 3′

most nucleotide of the first oligonucleotide is hybridizable to the sequence of interest.

19. The method of claim 16, wherein the second oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

20. The method of claim 16, wherein the 5′

most nucleotide of the second oligonucleotide is hybridizable to at least one nucleotide of the sequence of interest.

21. The method of claim 16, wherein the sequence of interest is a single nucleotide base.

22. The method of claim 16, wherein the sequence of interest is more than a single nucleotide base.

23. The method of claim 16, wherein the first and second oligonucleotide each comprise a sequence hybridizable to at least one nucleotide of the sequence of interest.

24. The method of claim 16, wherein the enzyme that cleaves RNA is RNase H.

25. The method of claim 16, wherein the target polynucleotide is DNA or cDNA.

26. The method of claim 16, wherein the target polynucleotide is amplified.

27. The method of claim 26, wherein amplification occurs simultaneously with generating multiple copies of a sequence of interest.

28. The method of claim 26, wherein amplification occurs prior to generating multiple copies of a sequence of interest.

29. The method of claim 16, wherein the target polynucleotide is attached to an analyte.

30. The method of claim 29, wherein the analyte is selected from the group consisting of a peptide, an antibody, an organic molecule and an inorganic molecule.

31. The method of claim 16, wherein said detectable identifying characteristic is selected from the group consisting of size of the cleaved attached oligonucleotide combination product, sequence of the cleaved attached oligonucleotide combination product, and detectable signal associated with the cleaved attached oligonucleotide combination product.

32. The method of claim 16, wherein the detectable identifying characteristic comprises the sequence of the cleaved attached oligonucleotide product, and wherein the sequence is detected by hybridizing the cleaved attached oligonucleotide product with a nucleic acid probe that is hybridizable to the cleaved attached oligonucleotide product.

33. The method of claim 32, wherein said nucleic acid probe comprises DNA.

34. The method of claim 32, wherein the nucleic acid probe is provided as an array.

35. The method of claim 34, wherein said array comprises the probe immobilized on a substrate fabricated from a material selected from the group consisting of paper, glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose, silicon, polystyrene, and optical fiber.

36. The method of claim 31, wherein, in step (b) the first oligonucleotide is extended, and wherein said detectable signal is associated with a label on a deoxyribonucleotide triphosphate or analog thereof that is incorporated during extension.

37. The method of claim 31, wherein said detectable signal is associated with interaction of two labels, wherein one label is located in the first oligonucleotide and a second label is located in the second oligonucleotide.

38. The method of claim 31, wherein said detectable signal is associated with interaction of two labels, wherein, in step (b) the first oligonucleotide is extended, and wherein one label is located on either the first or second oligonucleotide, and a second label is located on a deoxyribonucleotide triphosphate or analog thereof that is incorporated during extension.

39. The method of claim 16, wherein the attaching is covalent attachment.

40. The method of claim 39, wherein the covalent attachment is effected by ligase.

87. The method of claim 16, wherein the detection of the cleaved attached oligonucleotide combination product comprises directly detecting the detectable identifying characteristic of the cleaved attached oligonucleotide combination product.

88. The method of claim 16, wherein the cleaved attached oligonucleotide combination product is between 2 and 24 continuous nucleotides in length.

41. A method of linearly generating multiple copies of a nucleic acid sequence of interest from a polynucleotide template comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a target polynucleotide;

(b) a first oligonucleotide and a second oligonucleotide that hybridize to non-overlapping portions of a target polynucleotide, wherein the portion of the target polynucleotide that is hybridizable to the first oligonucleotide is 3′

with respect to the portion of the target nucleotide that is hybridizable to the second oligonucleotide, wherein at least one of said oligonucleotides is a composite primer comprising an RNA portion and a DNA portion such that (i) if said first oligonucleotide is the composite primer, said RNA portion is at the 5′

end of said first oligonucleotide, and (ii) if said second oligonucleotide is the composite primer, said RNA portion is at the 3′

end of said second oligonucleotide, and wherein at least one of said oligonucleotides comprises a sequence that is hybridizable to at least one nucleotide of the sequence of interest, and wherein said first or second oligonucleotide hybridizes only to said target polynucleotide and not to any dissociated amplification products thereof;

(c) optionally a DNA polymerase that lacks strand displacement activity;

(d) an enzyme that cleaves RNA from an RNA/DNA hybrid; and

(e) an agent that effects attachment of the first oligonucleotide and second oligonucleotide to each other when said oligonucleotides are hybridized to the target polynucleotide,

wherein the incubation is under conditions that permits oligonucleotide hybridization, optionally oligonucleotide extension, RNA cleavage and attachment of the first oligonucleotide and the second oligonucleotide, such that an attached oligonucleotide combination product is produced, and wherein the attached oligonucleotide combination product is of a size such that cleavage of RNA from the attached oligonucleotide combination product results in dissociation of the cleaved attached oligonucleotide product without strand displacement by an enzyme.

42. The method of claim 41, wherein the first oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

43. The method of claim 41, wherein the 3′

most nucleotide of the first oligonucleotide is hybridizable to the sequence of interest.

44. The method of claim 41, wherein the second oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

45. The method of claim 41, wherein the 5′

most nucleotide of the second oligonucleotide is hybridizable to at least one nucleotide of the sequence of interest.

46. The method of claim 41, wherein the sequence of interest is a single nucleotide base.

47. The method of claim 41, wherein the sequence of interest is more than a single nucleotide base.

48. The method of claim 41, wherein the first and second oligonucleotide each comprise a sequence hybridizable to at least one nucleotide of the sequence of interest.

49. The method of claim 41, wherein the enzyme that cleaves RNA is RNase H.

50. The method of claim 41, wherein the target polynucleotide is DNA or cDNA.

51. The method of claim 41, wherein the target polynucleotide is amplified.

52. The method of claim 51, wherein amplification occurs simultaneously with generating multiple copies of a sequence of interest.

53. The method of claim 51, wherein amplification occurs prior to generating multiple copies of a sequence of interest.

54. The method of claim 41, wherein the attaching is covalent.

55. The method of claim 54, wherein the covalent attachment is effected by ligase.

89. The method of claim 41, wherein the cleaved attached oligonucleotide product is between 2 and 24 continuous nucleotides in length.

56. A method of determining whether a nucleic acid sequence of interest is present or absent in a target polynucleotide comprising incubating a reaction mixture, said reaction mixture comprising:

(a) the target polynucleotide;

(b) a first oligonucleotide and a second oligonucleotide that hybridize to non-overlapping portions of the target polynucleotide, wherein the portion of the target polynucleotide that is hybridizable to the first oligonucleotide is 3′

with respect to the portion of the target nucleotide that is hybridizable to the second oligonucleotide, wherein at least one of said oligonucleotides is a composite primer comprising an RNA portion and a DNA portion such that (i) if said first oligonucleotide is the composite primer, said RNA portion is at the 5′

end of said first oligonucleotide, and (ii) if said second oligonucleotide is the composite primer, said RNA portion is at the 3′

end of said second oligonucleotide, and wherein at least one of said oligonucleotides comprises a sequence that is hybridizable to at least one nucleotide of the sequence of interest;

(c) optionally a DNA polymerase that lacks strand displacement activity;

(d) an enzyme that cleaves RNA from an RNA/DNA hybrid; and

(e) an agent that effects attachment of the first oligonucleotide and second oligonucleotide to each other when said oligonucleotides are hybridized to the target polynucleotide,

wherein the incubation is under conditions that permit oligonucleotide hybridization, optionally oligonucleotide extension, RNA cleavage and attachment of the first oligonucleotide and the second oligonucleotide, such that an attached oligonucleotide combination product comprising a detectable identifying characteristic is produced, and wherein the attached oligonucleotide combination product is of a size such that cleavage of the RNA from the attached oligonucleotide combination product results in dissociating of the cleaved attached oligonucleotide combination product from the target polynucleotide, without strand displacement by an enzyme and wherein said first or second oligonucleotide hybridize only to the target polynucleotide and not to said cleaved oligonucleotide combination product, whereby detection of the cleaved attached oligonucleotide combination product comprising the detectable identifying characteristic indicates presence of the nucleotide sequence of interest.

57. The method of claim 56, wherein the first oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

58. The method of claim 56, wherein the 3′

most nucleotide of the first oligonucleotide is hybridizable to the sequence of interest.

59. The method of claim 56, wherein the second oligonucleotide comprises a sequence hybridizable to at least one nucleotide of the sequence of interest.

60. The method of claim 56, wherein the 5′

most nucleotide of the second oligonucleotide is hybridizable to at least one nucleotide of the sequence of interest.

61. The method of claim 56, wherein the sequence of interest is a single nucleotide base.

62. The method of claim 56, wherein the sequence of interest is more than a single nucleotide base.

63. The method of claim 56, wherein the first and second oligonucleotide each comprise a sequence hybridizable to at least one nucleotide of the sequence of interest.

64. The method of claim 56, wherein the enzyme that cleaves RNA is RNase H.

65. The method of claim 56, wherein the DNA polymerase lacks strand displacement activity.

66. The method of claim 56, wherein the target polynucleotide is DNA or cDNA.

67. The method of claim 56, wherein the target polynucleotide is amplified.

68. The method of claim 67, wherein amplification occurs simultaneously with generating multiple copies of a sequence of interest.

69. The method of claim 67, wherein amplification occurs prior to generating multiple copies of a sequence of interest.

70. The method of claim 56, wherein the target polynucleotide is attached to an analyte.

71. The method of claim 70, wherein the analyte is selected from the group consisting of a peptide, an antibody, an organic molecule and an inorganic molecule.

72. The method of claim 56, wherein said detectable identifying characteristic is selected from the group consisting of size of the cleaved attached oligonucleotide combination product, sequence of the cleaved attached oligonucleotide combination product, and detectable signal associated with the cleaved attached oligonucleotide combination product.

73. The method of claim 56, wherein the detectable identifying characteristic comprises the sequence of the cleaved attached oligonucleotide product, and wherein the sequence is detected by hybridizing the cleaved attached oligonucleotide product with a nucleic acid probe that is hybridizable to the cleaved attached oligonucleotide product.

74. The method of claim 73, wherein said nucleic acid probe comprises DNA.

75. The method of claim 73, wherein the nucleic acid probe is immobilized on a surface.

76. The method of claim 75, wherein said surface comprises a substrate fabricated from a material selected from the group consisting of paper, glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose, silicon, polystyrene, and optical fiber.

77. The method of claim 72, wherein the incubation is under conditions that permit oligonucleotide extension, and wherein said detectable signal is associated with a label on a deoxyribonucleotide triphosphate or analog thereof that is incorporated during extension.

78. The method of claim 73, wherein said detectable signal is associated with interaction of two labels, wherein one label is located in the first oligonucleotide and a second label is located in the second oligonucleotide.

79. The method of claim 73, wherein said detectable signal is associated with interaction of two labels, wherein the incubation is under conditions that permit oligonucleotide extension, and wherein one label is located either the first or second oligonucleotide, and a second label is located on a deoxyribonucleotide triphosphate or analog thereof that is incorporated during extension.

80. The method of claim 56, wherein the attaching is covalent attachment.

81. The method of claim 80, wherein the covalent attachment is effected by ligase.

90. The method of claim 56, wherein detection of the cleaved attached oligonucleotide combination product comprises directly detecting the detectable identifying characteristic of the cleaved attached oligonucleotide combination product.

91. The method of claim 56, wherein the cleaved attached oligonucleotide combination product is between 2 and 24 continuous nucleotides in length.

82. The method of 16 or 56, wherein said method comprises determining whether two or more different sequences of interest are present or absent in a sample, said method using a two or more sets of first and second oligonucleotides, wherein the detectable identifying characteristics of the cleaved oligonucleotide attachment products corresponding to two or more different sequences of interest are different from each other.

83. The method of claim 82, wherein at least one of the sequences of interest is a variant of the sequence of interest.

84. A method of identifying an altered sequence of interest in a target polynucleotide, said method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) the target polynucleotide;

(b) a first oligonucleotide and a second oligonucleotide that hybridize to non-overlapping portions of the target polynucleotide, wherein the portion of the target polynucleotide that is hybridizable to the first oligonucleotide is 3′

with respect to the portion of the target nucleotide that is hybridizable to the second oligonucleotide, wherein at least one of said oligonucleotides is a composite primer comprising an RNA portion and a DNA portion such that (i) if said first oligonucleotide is the composite primer, said RNA portion is at the 5′

end of said first oligonucleotide, and (ii) if said second oligonucleotide is the composite primer, said RNA portion is at the 3′

end of said second oligonucleotide, and wherein at least one of said oligonucleotides comprises a sequence that is hybridizable to at least one nucleotide of the sequence of interest;

(c) optionally a DNA polymerase that lacks strand displacement activity;

(d) an enzyme that cleaves RNA from an RNA/DNA hybrid; and

(e) an agent that effects attachment of the first oligonucleotide and second oligonucleotide to each other when said oligonucleotides are hybridized to the target polynucleotide, wherein the incubation is under conditions that permit oligonucleotide hybridization, optionally oligonucleotide extension, RNA cleavage and attachment of the first oligonucleotide and the second oligonucleotide, such that an attached oligonucleotide combination product comprising a detectable identifying characteristic is produced,

and wherein the attached oligonucleotide combination product is of a size that when RNA is cleaved from the attached oligonucleotide combination product, the cleaved attached oligonucleotide combination product dissociates from the target polynucleotide, without strand displacement by an enzyme and wherein said first or second oligonucleotide hybridize only to the template polynucleotide and not to said cleaved oligonucleotide combination product, wherein production of detectably fewer cleaved oligonucleotide attachment products from the target as compared to the amount of cleaved oligonucleotide attachment products produced from a reference template comprising the sequence of interest indicates that the target polynucleotide contains an altered sequence of interest.

85. The method of claim 84, further comprising characterizing sequence of the target polynucleotide.

92. The method of claim 84, wherein the production of detectably fewer cleaved oligonucleotide attachment products is determined by directly detecting the detectable identifying characteristic of the cleaved oligonucleotide attachment products.

93. The method of claim 84, wherein the cleaved attached oligonucleotide combination product is between 2 and 24 nucleotides in length.