Methods and compositions for linear isothermal amplification of polynucleotide sequences, using a RNA-DNA composite primer

• US 6,251,639 B1
• Filed: 09/13/2000
• Issued: 06/26/2001
• Est. Priority Date: 09/13/1999
• Status: Expired due to Term

• Current Assignee: Elizabeth Davila, Juan Davila, NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US09/660,877
• Time in Patent Office: 286 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

The present invention provides novel isothermal, single primer linear nucleic acid amplification methods. Methods for amplifying complementary DNA using a composite primer, primer extension, strand displacement, and optionally a termination sequence, are provided. Methods for amplifying sense RNA using a composite primer, primer extension, strand displacement, optionally template switching, a propromoter oligonucleotide and transcription are also provided. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification products.

Claims

1. A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising:

(a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion;

(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′

with respect to hybridization of the composite primer to the template;

(c) extending the composite primer with DNA polymerase;

(d) cleaving the RNA portion of the annealed composite with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement,

whereby multiple copies of the complementary sequence of the target sequence are produced.

3. The method of claim 1 or 2, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

4. The method of claim 3, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

5. The method of claim 1 or 2, wherein a plurality of composite primers are used.

6. The method of claim 1 or 2, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oligonucleotide (TSO).

7. The method of claim 6, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.

8. The method of claim 1 or 2, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

9. The method of claim 8, wherein the blocking sequence comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocking sequence binds more tightly to the region as compared to a blocking sequence without the modification.

10. The method of claim 1 or 2, wherein the enzyme that cleaves RNA is RNaseH.

14. The method of claim 1 or 2, wherein steps (a) and (b) are performed in either order.

15. The method of claim 1 or 2, wherein steps (a) and (b) are performed simultaneously.

16. The method of claim 1 or 2, wherein steps (a), (b) and (c) are performed simultaneously.

17. The method of claim 1 or 2, wherein steps (a) and (b) are performed before step (c).

18. The method of claim 1 or 2, wherein all steps are performed simultaneously.

23. A method of characterizing a sequence of interest in a target polynucleotide, said method comprising

(i) amplifying a template polynucleotide sequence containing the sequence of interest by the method of claim 1 or 2, wherein the sequence of the RNA portion of the composite primer is known, and

(ii) comparing the amplification products if any from step (i) with the amount of amplification products from a reference template

wherein

(1) production of detectably fewer amplification products from the template as compared to the amount of amplification products from the reference template which comprises a region complementary to the RNA portion of the composite primer indicates that the target polynucleotide does not comprise a sequence complementary to the RNA portion of the composite primer and is a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer; or

(2) production of detectably more amplification products from the template as compared to the amount of amplification products from the reference template which does not comprise a region which is complementary to the RNA portion of the composite primer indicates that the target polynucleotide comprises a sequence complementary to the RNA portion of the composite primer and is not a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer.

24. The method of claim 23, wherein the sequence of the RNA portion of the composite primer comprises a sequence complementary to a wild type sequence, and the sequence of interest is characterized in determining the presence or absence of the wild type sequence.

25. The method of claim 23, wherein the sequence of the RNA portion of the composite primer comprises a sequence complementary to a mutant sequence, and the sequence of interest is characterized in determining the presence or absence of the mutant sequence.

26. The method of claim 23, wherein the sequence of the RNA portion of the composite primer comprises a sequence complementary to an allelic sequence, and the sequence of interest is characterized in determining the presence or absence of the allelic sequence.

27. A method of detecting a mutation in a target polynucleotide by single stranded conformation polymorphism, comprising (i) amplifying the target polynucleotide by the method of claim 1 or 2;

and (ii) analyzing the amplification products for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide.

28. The method of claim 23, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

29. The method of claim 28, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

30. A method of producing a microarray, comprising (i) amplifying a polynucleotide sequence by the method of claim 1 or 2;

and (ii) attaching the amplified products onto a solid substrate to make a microarray of the amplified products.

72. The method of claim 23, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

73. The method of claim 72, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

74. The method of claim 23, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oliognucleotide (TSO).

75. The method of claim 74, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.

76. The method of claim 23, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

77. The method of claim 76, wherein the blocking sequence comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocking sequence binds more tightly to the region as compared to a blocking sequence without the modification.

78. The method of claim 23, wherein the enzyme that cleaves RNA is RNaseH.

80. The method of claim 23, wherein the polynucleotide comprising the propromoter comprises a region at the 3′

end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.

81. The method of claim 80, wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).

82. The method of claim 23, wherein steps (a) and (b) are performed in either order.

83. The method of claim 23, wherein steps (a) and (b) are performed simultaneously.

84. The method of claim 23, wherein steps (a), (b) and (c) are performed simultaneously.

85. The method of claim 23, wherein steps (a) and (b) are performed before step (c).

86. The method of claim 23, wherein all steps except the step of comparing the amplification products are performed simultaneously.

87. The method of claim 27, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oliognucleotide (TSO).

88. The method of claim 87, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.

89. The method of claim 27, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

90. The method of claim 89, wherein the blocking sequence comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocking sequence binds more tightly to the region as compared to a blocking sequence without the modification.

91. The method of claim 27, wherein the enzyme that cleaves RNA is RNaseH.

93. The method of claim 27, wherein the polynucleotide comprising the propromoter comprises a region at the 3′

end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.

94. The method of claim 93, wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).

95. The method of claim 27, wherein steps (a) and (b) are performed in either order.

96. The method of claim 27, wherein steps(a) and (b) are performed simultaneously.

97. The method of claim 27, wherein steps (a), (b) and (c) are performed simultaneously.

98. The method of claim 27, wherein steps (a) and (b) are performed before step (c).

99. The method of claim 27, wherein all steps except the step of analyzing the amplification products are performed simultaneously.

100. The method of claim 27, wherein the mutation is selected from the group consisting of a base substitution, a base insertion, a base deletion and a single nucleotide polymorphism.

101. The method of claim 27, wherein said step of analyzing the amplification products comprises a method selected from the group consisting of gel electrophoresis, capillary electrophoresis and cleavage of the amplification products using nucleases which recognize sequence dependent secondary structures.

102. The method of claim 30, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oliognucleotide (TSO).

103. The method of claim 102, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.

104. The method of claim 30, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

105. The method of claim 104, wherein the blocking sequence comprise a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocking sequence binds more tightly to the region as compared to a blocking sequence without the modification.

106. The method of claim 30, wherein the enzyme that cleaves RNA is RNaseH.

107. The method of claim 30, wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).

110. The method of claim 30, wherein steps (a) and (b) are performed in either order.

111. The method of claim 30, wherein steps (a) and (b) are performed simultaneously.

112. The method of claim 30, wherein steps (a), (b) and (c) are performed simultaneously.

113. The method of claim 30, wherein steps (a) and (b) are performed before step (c).

114. The method of claim 30, wherein all steps except the step of attaching the amplified products are performed simultaneously.

2. A method for amplifying a target polynucleotide sequence comprising:

(a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion;

(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′

with respect to hybridization of the composite primer to the template;

(c) extending the composite primer with DNA polymerase;

(d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement to produce displaced primer extension product;

(e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products,

whereby multiple copies of the target sequence are produced.

11. The method of claim 2, wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).

12. The method of claim 2, wherein the polynucleotide comprising the propromoter comprises a region at the 3′

end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.

13. The method of claim 12, wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).

79. A method of characterizing a sequence of interest in a target polynucleotide, said method comprising

(i) amplifying a template polynucleotide sequence containing the sequence of interest by the method of claim 2, wherein the sequence of the RNA portion of the composite primer is known, and

(ii) comparing the amplification products if any from step (i) with the amount of amplification products from a reference template

wherein (1) production of detectably fewer amplification products from the template as compared to the amount of amplification products from the reference template which comprises a region complementary to the RNA portion of the composite primer indicates that the target polynucleotide does not comprise a sequence complementary to the RNA portion of the composite primer and is a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer; or

(2) production of detectably more amplification products from the template as compared to the amount of amplification products from the reference template which does not comprise a region which is complementary to the RNA portion of the composite primer indicates that the target polynucleotide comprises a sequence complementary to the RNA portion of the composite primer and is not a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer.

and wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).

92. A method of detecting a mutation in a target polynucleotide by single stranded conformation polymorphism comprising (i) amplifying the target polynucleotide by the method of claim 2 and (ii) analyzing the amplification products for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide

and wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).

108. A method of producing a microarrray, comprising (i) amplifying a polynucleotide sequence by the method of claim 2;

and (ii) attaching the amplified products onto a solid substrate to make a microarray of the amplified products

and wherein the polynucleotide comprising the propromoter comprises a region at the 3′

end which hybridizes to the displaced primer on product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.

109. The method of claim 108, wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).

19. A method of sequencing a target nucleotide sequence comprising:

(a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion;

(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′

with respect to hybridization of the composite primer to the template;

(c) extending the composite primer with DNA polymerase and a mixture of dNTPs and dNTP analogs, such that primer extension is terminated upon incorporation of a dNTP analog;

(d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced of varying lengths;

(e) analyzing the product of steps (a) through (d) to determine sequence.

21. The method of claim 19 or 20, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

22. The method of claim 21, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

59. The method of claim 19 or 20, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oligonucleotide (TSO).

60. The method of claim 59, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.

61. The method of claim 19 or 20, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

62. The method of claim 61, wherein the blocking sequence comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the blocking sequence binds more tightly to the region as compared to a blocking sequence without the modification.

63. The method of claim 19 or 20, wherein the enzyme that cleaves RNA is RNaseH.

67. The method of claim 19 or 20, wherein steps (a) and (b) are performed in either order.

68. The method of claim 19 or 20, wherein steps (a) and (b) are performed simultaneously.

69. The method of claim 19 or 20, wherein steps (a), (b) and (c) are performed simultaneously.

70. The method of claim 19 or 20, wherein steps (a) and (b) are performed before step (c).

71. The method of claim 19 or 20, wherein all steps except the step of analyzing the product are performed simultaneously.

20. A method of sequencing a target nucleotide sequence comprising

(a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion;

(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′

with respect to hybridization of the composite primer to the template;

(c) extending the composite primer with DNA polymerase;

(d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement to produce displaced primer extension product;

(e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions such that transcription occurs from the extension product by RNA polymerase, using a mixture of rNTPs and rNTP analogs, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products, and such that transcription is terminated upon incorporation of an rNTP analog, whereby multiple copies of the target sequence are produced of varying lengths;

(f) analyzing the product of steps (a) through (e) to determine sequence.

64. The method of claim 20, wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).

65. The method of claim 20, wherein the polynucleotide comprising the propromoter comprises a region at the 3′

end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.

66. The method of claim 65, wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).

31. A composition comprising (a) a composite primer, said composite primer comprising a 3′

DNA portion and a 5′

RNA portion, and (b) a polynucleotide comprising a termination polynucleotide sequence, wherein the termination polynucleotide sequence effects cessation of DNA replication of a template by DNA polymerase.

32. The composition of claim 31, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

33. The composition of claim 31, wherein the 5′

RNA portion is about 5 to about 20 nucleotides and the 3′

DNA portion is about 5 to about 15 nucleotides.

34. The composition of claim 31, wherein the polynucleotide comprising a termination polynucleotide sequence is a TSO.

35. The composition of claim 34, wherein the TSO comprises a modification in the region which hybridizes to the template, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.

36. The composition of claim 31 wherein the termination polynucleotide sequence is a blocking sequence.

37. The composition of claim 31, further comprising a propromoter template oligonucleotide (PTO).

38. A composition comprising a complex of (a) a template strand;

(b) a composite primer, said composite primer comprising a 3′

DNA portion and an RNA portion; and

(c) a polynucleotide comprising a termination polynucleotide sequence, wherein the termination polynucleotide sequence effects cessation of DNA replication of a template by DNA polymerase.

39. The composition of claim 38, wherein the RNA portion is 5′

and adjacent to the 3′

DNA portion.

40. The composition of claim 38, wherein the polynucleotide comprising a termination polynucleotide sequence is a TSO.

41. The composition of claim 38 wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

42. A reaction mixture comprising (a) a polynucleotide template;

(b) a composite primer comprising a 3′

DNA portion and an RNA portion;

(c) a DNA polymerase; and

(d) a polynucleotide comprising a termination polynucleotide sequence, wherein the termination polynucleotide sequence effects cessation of DNA replication of a template by DNA polymerase.

43. The reaction mixture of claim 42, wherein the composite primer comprises a 5′

RNA portion which is adjacent to the 3′

DNA portion.

44. The reaction mixture of claim 43, further comprising an enzyme which cleaves RNA from an RNA/DNA hybrid.

45. The reaction mixture of claim 44, wherein the enzyme is RNaseH.

46. The reaction mixture of claim 42, further comprising a polynucleotide comprising a propromoter.

47. The reaction mixture of claim 46, wherein the polynucleotide comprising a propromoter is a TSO.

48. The reaction mixture of claim 46, wherein the polynucleotide comprising a propromoter is a propromoter template oligonucleotide (PTO).

115. The reaction mixture of claim 42, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oligonucleotide.

116. The reaction mixture of claim 42, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

49. A kit for amplification of a target polynucleotide sequence, comprising (a) a composite primer comprising a 3′

DNA portion and an RNA portion, and (b) a polynucleotide comprising a termination polynucleotide sequence, wherein the termination polynucleotide sequence effects cessation of DNA replication of a template by DNA polymerase.

50. The kit of claim 49, wherein the RNA portion is 5′

with respect to the 3′

DNA portion.

51. The kit of claim 50, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

52. The kit of claim 49, wherein the polynucleotide comprising a termination polynucleotide sequence is a TSO.

53. The kit of claim 49, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocker sequence.

54. The kit of claim 49, further comprising a polynucleotide comprising a propromoter.

55. The kit of claim 54, wherein the polynucleotide comprising a propromoter is a TSO.

56. The kit of claim 54, wherein the polynucleotide comprising a propromoter is a PTO.

57. The kit of claim 49, further comprising an enzyme which cleaves RNA from an RNA/DNA hybrid.

58. The kit of claim 57, wherein the enzyme is RNaseH.