Methods and compositions for linear isothermal amplification of polynucleotide sequences

• US 6,692,918 B2
• Filed: 11/21/2001
• Issued: 02/17/2004
• 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): Siew, Jeffrey
• Application Number:
  US09/990,531
• Publication Number:
  US 20030017591A1
• Time in Patent Office: 818 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24-- 2
• US Class Current: 435/6.1
• 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 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) extending a composite primer in a complex comprising (i) a DNA template strand comprising the target sequence; and

(ii) the composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion, wherein the DNA template strand is hybridized to the composite primer;

(b) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such tat 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.

4. A method for amplifying a target polynucleotide sequence comprising generating displaced primer extension product using the method of any of claims 1-3, further comprising:

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.

7. The method of claim 4, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

9. The method of claim 7, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

14. The method of claim 7, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

17. The method of claim 4, wherein the enzyme that cleaves RNA is RNaseH.

19. The method of claim 4, wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).

21. The method of claim 4, wherein the polynucleotide comprising the propromoter comprises a region 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.

23. The method of claim 21, wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).

25. The method of claim 4, wherein the polynucleotide comprising the propromoter comprises:

(a) a termination polynucleotide sequence that does not effect template switch under conditions wherein the termination polynucleotide sequence is hybridizable to the DNA template;

(b) a propromoter sequence, wherein the propromoter sequence is not hybridizable to the DNA template under conditions wherein the termination polynucleotide sequence is hybridizable to the DNA template; and

(c) a sequence which is hybridizable to a complementary sequence of the target polynucleotide.

46. The method of claim 9, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 5 to about 20 nucleotides.

48. The method of claim 9, wherein the RNA portion of the composite primer consists of about 10 to about 20 nucleotides and the DNA portion of the composite primer consists of about 7 to about 20 nucleotides.

58. The method of claim 9, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 3 to about 20 nucleotides.

60. The method of any of claims 5, 21-30, or 42, wherein the method comprises 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.

97. The method of any of claims 1, 3, 5, 27-30, 44, 45, 63 or 64, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

98. The method of claim 97, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

99. The method of any of claims 1, 3, 5, 27-30, 44, 45, 63 or 64, wherein the enzyme that cleaves RNA is RNaseH.

100. The method of claim 98, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 5 to about 20 nucleotides.

101. The method of claim 98, wherein the RNA portion of the composite primer consists of about 10 to about 20 nucleotides and the DNA portion of the composite primer consists of about 7 to about 20 nucleotides.

102. The method of claim 98, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 3 to about 20 nucleotides.

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

(a) extending a composite primer in a complex comprising (i) a DNA template strand comprising the target sequence;

(ii) the composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion, wherein the composite primer is hybridized to the DNA template strand; and

(iii) a polynucleotide comprising a termination polynucleotide sequence, wherein the polynucleotide comprising a termination polynucleotide sequence is hybridized to a region of the template which is 5′

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

(b) 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.

11. The method of claim 2 or 5, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oligonucleotide (TSO).

13. The method of claim 2 or 5, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

15. The method of claim 2 or 5, wherein the termination polynucleotide sequence does not effect template switch under conditions wherein the termination polynucleotide sequence is hybridizable to the DNA template, and wherein the polynucleotide comprising the termination polynucleotide sequence further comprises (a) a propromoter sequence, wherein to propromoter sequence is not hybridizable to the DNA template under conditions wherein the termination polynucleotide sequence is hybridizable to the DNA template;

and (b) a sequence which is hybridizable to a complementary sequence of the target polynucleotide sequence.

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

cleaving an RNA portion of a composite primer extension product in a complex comprising (a) a DNA template strand comprising the target sequence; and

(b) the composite primer extension product, wherein the composite primer of the composite primer extension product comprises an RNA portion and a 3′

DNA portion, wherein the composite primer extension product is hybridized to the DNA template strand;

wherein said cleaving is with an enzyme that cleaves RNA from an RNA/DNA hybrid, whereby 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.

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

with respect to the 3′

DNA portion.

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

RNA portion is adjacent to the 3′

DNA portion.

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

hybridizing a primer extension product with a polynucleotide comprising a propromoter and a region which hybridizes to the primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the primer extension product, wherein the primer extension product is a displaced primer extension product generated by;

(a) hybridizing a DNA template strand 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;

whereby multiple copies of the target sequence are produced.

20. The method of claim 5, wherein the polynucleotide comprising a propromoter and region which hybridizes to the displaced primer extension product is a template switch oligonucleotide (TSO).

22. The method of claim 5, wherein the polynucleotide comprising the propromoter comprises a region 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.

24. The method of claim 22, wherein the polynucleotide comprising the propromoter is a propromoter template oligonucleotide (PTO).

26. The method of claim 5, wherein the polynucleotide comprising the propromoter comprises:

(a) a termination polynucleotide sequence that does not effect template switch under conditions wherein the termination polynucleotide sequence is hybridizable to the DNA template;

(b) a propromoter sequence, wherein the propromoter sequence is not hybridizable to the DNA template under conditions wherein the termination polynucleotide sequence is hybridizable to the DNA template; and

(c) a sequence which is hybridizable to a complementary sequence of the target polynucleotide.

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

(a) hybridizing a first composite primer to a DNA template strand comprising the complementary sequence of the target sequence, said composite primer comprising an RNA portion and a 3′

DNA portion, wherein said DNA template strand is generated by a method comprising;

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

DNA portion;

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

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

(iii) extending the second composite primer with DNA polymerase;

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

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

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

(c) extending the first composite primer wit DNA polymerase;

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

whereby multiple copies of the target sequence are produced.

8. The method of claim 6, wherein the RNA portion of the first composite primer and the second composite primer is 5′

with respect to the 3′

DNA portion.

10. The method of claim 8, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

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

16. The method of claim 6, wherein the termination polynucleotide sequence does not effect template switch under conditions wherein the termination polynucleotide sequence is hybridizable to the DNA template, and wherein the polynucleotide comprising the termination polynucleotide sequence further comprises (a) a propromoter sequence, wherein the propromoter sequence is not hybridizable to the DNA template under conditions wherein the termination sequence is hybridizable to the DNA template;

and (b) a sequence which is hybridizable to a complementary sequence of the target polynucleotide sequence.

18. The method of claim 6, wherein the enzyme that cleaves RNA is RNaseH.

47. The method of claim 10, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 5 to about 20 nucleotides.

49. The method of claim 10, wherein the RNA portion of the composite primer consists of about 10 to about 20 nucleotides and the DNA portion of the composite primer consists of about 7 to about 20 nucleotides.

59. The method of claim 10, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 3 to about 20 nucleotides.

61. The method of claim 6, wherein the method comprises hybridizing a second polynucleotide comprising a termination polynucleotide sequence to a region of the template comprising the target sequence which is 5′

with respect to hybridization of the second composite primer to said template.

62. The method of claim 6, wherein the method comprises hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template comprising the complement of the target sequence which is 5′

with respect to hybridization of the first composite primer to the template.

27. A method of sequencing a target polynucleotide sequence comprising sequencing a nucleic acid amplification product, wherein said nucleic acid amplification product is generated by a method comprising:

(i) hybridizing DNA template strand comprising the complement of the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion;

(ii) 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 said template;

(iii) extending the composite primer with DNA polymerase;

(iv) 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 nucleic acid amplification products comprising the target sequence are generated.

28. A method of sequencing a target polynucleotide sequence comprising sequencing a nucleic arid amplification product, wherein said nucleic acid amplification product is generated by a method comprising:

(i) hybridizing a DNA template strand comprising the target sequence wit a composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion;

(ii) 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 said template;

(iii) extending the composite primer with DNA polymerase;

(iv) 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 nucleic acid amplification products comprising the complement of the target sequence are generated.

29. A method of detecting whether a target polynucleotide sequence is present in a sample, said method comprising detecting whether the target sequence is present in a nucleic acid amplification product, wherein said nucleic acid amplification product is generated by:

(i) hybridizing a DNA template strand comprising the complement of the target polynucleotide with a composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion;

(ii) 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 said template;

(iii) extending the composite primer with DNA polymerase;

(iv) 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 nucleic acid amplification products comprising the target sequence are generated.

31. The method of claim 29 or 30, wherein said target sequence comprises a mutation.

33. The method of claim 29, wherein the target sequence is detected by hybridizing said amplification product with a nucleic acid probe that is hybridizable to said target sequence.

35. The method of claim 33 or 34, wherein said nucleic acid probe comprises DNA.

36. The method of claim 33 or 34, wherein said nucleic acid probe comprises RNA.

37. The method of claim 33 or 34, wherein said nucleic acid probe comprises RNA and DNA.

38. The method of claim 33 or 34, wherein said nucleic acid probe comprises peptide nucleic acid (PNA).

39. The method of claim 33 or 34, or wherein said probe is provided as a microarray.

40. The method of claim 39, wherein said microarray 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 and optical fiber.

41. The method of claim 29, wherein the target sequence is detected by limited primer extension.

30. A method of detecting whether a target polynucleotide sequence is present in a sample, said method comprising detecting whether the target sequence is present in a nucleic acid amplification product, wherein said nucleic acid amplification product is generated by:

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

DNA portion;

(ii) 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 said template;

(iii) extending the composite primer with DNA polymerase;

(iv) 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 nucleic acid amplification products comprising the complement of the target sequences are generated.

34. The method of claim 30, wherein the target sequence is detected by hybridizing said amplification product with a nucleic acid probe that is hybridizable to the complement of the target sequence.

32. The method of 31, wherein said mutation is a single nucleotide polymorphism.

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

(a) hybridizing a DNA template strand 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 tat 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 tat RNA transcripts are produced comprising sequences complementary to the displaced primer extension products,

whereby at least 100 copies of RNA transcripts are produced from each displaced primer extension product.

43. The method of claim 42, wherein between 100 to 1000 copies of RNA transcripts are produced from each displaced primer extension product.

44. A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a DNA template strand;

(b) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises an RNA portion and a 3′

DNA portion;

(c) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are generated.

104. The method of claim 44, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

107. The method of claim 44, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

45. A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex comprising (i) a DNA template strand, and (ii) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises an RNA portion and a 3′

DNA portion;

(b) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are generated.

105. The method of claim 45, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

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

RNA portion is adjacent to the 3′

DNA portion.

50. A kit for amplifying a polynucleotide sequence comprising a composite primer, wherein said composite primer comprises an RNA portion and a 3′

DNA portion, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion, and wherein the RNA portion of the composite primer consists of about 5 to about 20 nucleotides and the DNA portion of the composite primer consists of about 3 to about 12 nucleotides.

51. The kit of claim 50, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides.

52. The kit of claim 50, wherein the RNA portion of the composite primer consists of about 7 to about 18 nucleotides.

53. The kit of any of claims 50-52, wherein the DNA portion of the composite primer consists of about 5 to about 12 nucleotides.

54. The kit of any of claims 50-52, wherein the DNA portion of the composite primer consists of about 7 to about 12 nucleotides.

55. The kit of claim 50, further comprising an enzyme that cleaves RNA from an RNA/DNA hybrid.

56. The kit of claim 55, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNaseH.

57. The kit of claim 56, further comprising a DNA polymerase.

96. The kit of claim 50, wherein the composite primer consists of the 5′

RNA portion and the 3′

DNA portion.

109. The kit of claim 50, further comprising a set of instructions for carrying out a method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex comprising (i) a DNA template strand, and (ii) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises a 5′

RNA portion and a 3′

DNA portion;

(b) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement.

63. A method of detecting whether a target polynucleotide sequence is present in a sample, said method comprising detecting whether the target sequence is present in a nucleic acid amplification product, wherein said nucleic acid amplification product is generated by:

(a) extending a composite primer in a complex comprising (i) a DNA template strand comprising the complement of the target sequence; and

(ii) the composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion, wherein the DNA template strand is hybridized to the composite primer;

(b) 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 nucleic acid amplification products comprising the target sequence are generated.

65. The method of claim 63 or 64, wherein said target sequence comprises a mutation.

67. The method of claim 63, wherein the target sequence is detected by hybridizing sad amplification product with a nucleic acid probe that is hybridizable to said target sequence.

69. The method of claim 67 or 68, wherein said nucleic acid probe comprises DNA.

70. The method of claim 67 or 68, wherein said nucleic acid probe comprises RNA.

71. The method of claim 67 or 68, wherein said nucleic acid probe comprises RNA and DNA.

72. The method of claim 67 or 68, wherein said nucleic acid probe comprises peptide nucleic acid (PNA).

73. The method of claim 67 or 68, wherein said probe is provided as a microarray.

74. The method of claim 73, wherein said microarray comprises the probe immobilized a substrate fabricated from a material selected from the group consisting of paper, glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose, silicon, and optical fiber.

75. The method of claim 63, wherein the target sequence is detected by limited primer extension.

64. A method of detecting whether a target polynucleotide sequence is present in a sample, said method comprising detecting whether the target sequence is present in a nucleic acid amplification product, wherein said nucleic acid amplification product is generated by:

(a) extending a composite primer in a complex comprising (i) a DNA template strand comprising the target sequence; and

(ii) the composite primer, said composite primer comprising an RNA portion and a 3′

DNA portion, wherein the DNA template strand is hybridized to the composite primer;

(b) 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 stand displacement,

whereby nucleic amplification products comprising the complement of the target sequence are generated.

68. The method of claim 64, wherein the target sequence is detected by hybridizing said amplification product wit a nucleic acid probe that is hybridizable to the complement of the target sequence.

76. The method of claim 64, wherein the target sequence is detected by limited primer extension.

66. The method of 65, wherein said mutation is a single nucleotide polymorphism.

77. A kit for amplifying a polynucleotide sequence, comprising a composite primer, wherein the composite primer comprises an RNA portion and a 3′

DNA portion, and wherein the RNA portion of the composite primer consists of about 5 to about 25 nucleotides and the DNA portion of the composite primer consists of about 3 to about 12 nucleotides.

110. The kit of claim 77, further comprising a set of instructions for carrying out a method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex comprising (i) a DNA template strand, and (ii) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises a 5′

RNA portion and a 3′

DNA portion;

(b) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement.

78. A kit for amplifying a polynucleotide sequence, comprising a composite primer, wherein the composite primer comprises a 5′

RNA portion and a 3′

DNA portion, and wherein the RNA portion of the composite primer consists of about 3 to about 25 nucleotides and the DNA portion of the composite primer consists of about 3 to about 12 nucleotides.

79. The kit of claim 78, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

81. The kit of claim 78, wherein the RNA portion of the composite primer consists of about 7 to about 20 nucleotides.

84. The kit of claim 78, wherein the RNA portion of the composite primer consists of about 10 to about 20 nucleotides.

85. The kit of claim 84, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

86. The kit of any of claims 78-85, wherein the DNA portion of the composite primer consists of about 5 to about 12 nucleotides.

87. The kit of any of claims 78-85, wherein the DNA portion of the composite primer consists of about 7 to about 12 nucleotides.

88. The kit of claim 78, further comprising an enzyme that cleaves RNA from an RNA/DNA hybrid.

89. The kit of claim 88, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNaseH.

90. The kit of claim 88, further comprising a DNA polymerase.

111. The kit of claim 78, further comprising a set of instructions for carrying out a method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex comprising (i) a DNA template strand, and (ii) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises a 5′

RNA portion and a 3′

DNA portion;

(b) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement.

112. The kit of claim 79, further comprising a set of instructions for carrying out a method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex comprising (i) a DNA template strand, and (ii) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises a 5′

RNA portion and a 3′

DNA portion;

(b) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement.

80. The kit of to claim wherein the composite primer consists of the 5′

RNA portion and the 3′

DNA portion.

82. The kit of claim the RNA portion of the composite primer consists of about 7 to about 18 nucleotides.

83. The kit of claim 82, wherein the composite primer consists of the 5′

RNA portion and the 3′

DNA portion.

91. A kit for amplifying a polynucleotide sequence comprising a composite primer, wherein said composite primer comprises a 5′

RNA portion and a 3′

DNA portion, wherein the RNA portion of the composite primer consists of about 10 to about 20 nucleotides and the DNA portion of the composite primer consists of about 7 to about 12 nucleotides;

a DNA polymerase; and

an enzyme that cleaves RNA from a RNA/DNA hybrid.

92. The kit of claim 91, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

93. The kit of claim 91, wherein the composite primer consists of the 5′

RNA portion and the 3′

DNA portion.

94. The kit of claim 92, wherein the composite primer consists of the 5′

RNA portion and the 3′

DNA portion.

95. The kit of claim 91, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNaseH.

113. The kit of claim 91, further comprising a set of instructions for carrying out a method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex comprising (i) a DNA template strand, and (ii) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises a 5′

RNA portion and a 3′

DNA portion;

(b) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement.

114. The kit of claim 92, further comprising a set of instructions for carrying out a method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex comprising (i) a DNA template strand, and (ii) a composite primer that is hybridizable to the DNA template strand, wherein the composite primer comprises a 5′

RNA portion and a 3′

DNA portion;

(b) a DNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the primer extension product from the template when RNA is cleaved from the primer extension product whereby another composite primer hybridizes and repeats primer extension by strand displacement.