Methods and compositions for amplification of RNA sequences

• US 7,094,536 B2
• Filed: 03/11/2002
• Issued: 08/22/2006
• Est. Priority Date: 03/09/2001
• 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): HORLICK, KENNETH R
• Application Number:
  US10/096,221
• Publication Number:
  US 20020164628A1
• Time in Patent Office: 1,625 Days
• Field of Search: 435/91.1, 435/91.2, 435/810, 536/23.1, 536/24.33
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• 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 2525/143 incorporating a promoter sequence

Abstract

The invention provides methods for linear and exponential amplification of RNA. They are particularly suitable for amplifying a plurality of RNA species in a sample. The methods are based on hybridization of polynucleotide comprising a propromoter sequence to a primer extension product to generate an intermediate polynucleotide capable of driving transcription, whereby multiple copies of RNA products comprising sequences complementary to an RNA sequence of interest are generated. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

Claims

1. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising the steps of:

(a) extending a first primer hybridized to a target RNA with an RNA-dependent DNA polymerase, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) cleaving RNA in the complex of step (a) with an enzyme that cleaves RNA from an RNA/DNA hybrid;

(c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase, whereby a complex comprising the first primer extension product and a second primer extension product is produced;

(d) denaturing the complex of step (c); and

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

whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

3. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a single stranded second primer extension product resulting from step (d) of claim 1;

(b) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to the single stranded second primer extension product; and

an RNA polymerase;

wherein the incubation is under conditions that permit propromoter polynucleotide hybridization and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

4. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a single stranded second primer extension product resulting from step (d) of claim 1;

(b) an exponential amplification primer comprising a sequence hybridizable to an RNA transcript comprising a sequence complementary to the target RNA;

(c) a first propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded second primer extension product;

(d) a second propromoter polynueleotide comprising a propromoter and a region which is hybridizable to a single stranded exponential amplification extension product;

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

(f) an RNA polymerase;

wherein the incubation is under conditions that permit primer extension, RNA cleavage, propromoter polynucleotide hybridization and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

5. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) an RNA transcript from step (e) of claim 1;

(b) an exponential amplification primer comprising a sequence hybridizable to the RNA transcript;

(c) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded exponential amplification primer extension product;

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

(e) an RNA polymerase;

wherein the incubation is under conditions that permit primer extension, RNA cleavage, propromoter polynucleotide hybridization and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

14. The method of claim 1 or 2, wherein the target RNA is mRNA.

15. The method of claim 1 or 2, wherein the first primer comprises a poly-T sequence.

20. The method of claim 1 or 2, wherein at least one type of rNTP used is a labeled rNTP, whereby labeled products are generated.

21. The method of claim 1 or 2, wherein the first primer is a random primer.

22. The method of claim 1 or 2, wherein the second primer comprises DNA.

23. The method of claim 1 or 2, wherein the second primer comprises a fragment of the target RNA hybridized to the primer extension product, said fragment generated by cleaving RNA in the complex of step (b) with an enzyme that cleaves RNA from an RNA/DNA hybrid.

24. The method of claims 1 or 2, wherein said method comprises generating multiple copies of two or more different sequences of interest.

25. The method of claim 24, wherein said method comprises at least two different first primers.

32. The method of claims 1, 2, 4, 5, 6, 7, 8 and 9, wherein the enzyme that cleaves RNA is RNase H.

33. The method of any of claims 1, 2, 6, 7, 8, and 9, wherein the RNA-dependent DNA polymerase and DNA-dependent DNA polymerase are one enzyme.

34. The method of any of claims 1, 2, 6, 7, 8, and 9, wherein the RNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

35. The method of any of claims 1, 2, 6, 7, 8, and 9, wherein the DNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

36. The method of any of claims 1, 2, 6, 7, 8, and 9, wherein the DNA-dependent DNA polymerase, the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

37. The method of any of claims 1, 6, and 8, wherein the propromoter polynucleotide comprises a region at the 3′

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

38. The method of claim 37, wherein the propromoter polynucleotide is a propromoter template oligonucleotide.

42. A method of sequencing an RNA sequence of interest, said method comprising analyzing amplification products to determine sequence, said amplification products produced by the method of any of claims 1, 6, 7, and 10 in the presence of a mixture of rNTPs and rNTP analogs such that transcription is terminated upon incorporation of an rNTP analog.

43. The method of claim 42, wherein the target RNA is mRNA.

46. A method of detecting a mutation in a target RNA, comprising analyzing sequences of amplification products for the presence of a mutation as compared to a reference polynucleotide sequence, said amplification products produced by the method of any of claims 1, 2, 6, 7, 8, 9, and 10.

47. The method of claim 46, wherein the target RNA is mRNA.

48. A method of detecting a mutation in a target RNA by single stranded conformation polymorphism, comprising analyzing amplification products for single stranded conformation, said amplification products produced by the method of any of claims 1, 2, 6, 7, 8, 9, 10, and 11, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide.

49. The method of claim 48, wherein the target RNA is mRNA.

50. A method of producing a nucleic acid immobilized to a substrate comprising immobilizing amplification products on a substrate, said amplification products produced by the method of any of claims 1, 2, 6, 7, 8, 9, 10, and 11.

51. The method of claim 50, wherein the target RNA is mRNA.

52. The method of claim 50, wherein the substrate is a microarray.

53. A method of characterizing an RNA sequence of interest, comprising analyzing labeled RNA products, said labeled RNA products produced by amplifying a target RNA by the method of claim 20.

54. The method of claim 53, wherein analyzing comprises contacting the labeled RNA products with at least one probe sequence.

55. The method of claim 54, wherein the at least one probe sequence is provided as a microarray.

56. The method of claim 55, wherein the microarray comprises at least one probe immobilized on a substrate fabricated from a material selected from the group consisting of paper, ceramic, glass, plastic, polypropylene, polystyrene, nylon, polyacrylamide, nitrocellulose, silicon, and optical fiber.

57. The method of claim 56, wherein the probe is immobilized on the substrate in a two-dimensional configuration or a three-dimensional configuration comprising pins, rods, fibers, tapes, threads, beads, particles, microtiter wells, capillaries, and cylinders.

58. The method of claim 53, wherein the target RNA is mRNA.

59. The method of claim 53, wherein analyzing the labeled RNA products comprises determining amount of said products, whereby the amount of the RNA sequence of interest present in a sample is quantified.

67. A method of determining gene expression profile in a sample, said method comprising determining amounts of amplification products of each of at least two RNA sequences of interest, said amplification products amplified by the method of any of claims 1, 2, 6, 7, 8, 9, 10, and 11, wherein each said amount is indicative of amount of each RNA sequence of interest in the sample, whereby the gene expression profile in the sample is determined.

68. The method of claim 67, wherein each target RNA is mRNA.

69. A method of preparing a library, said method comprising:

preparing a library from amplified single stranded DNA or RNA product, said amplified single stranded DNA or RNA product amplified by amplifying at least two RNA sequences of interest using the method of any one of claims 1, 2, 6, 7, 8, 9, 10, and 11.

70. The method of claim 69, wherein the first primer is a random primer.

71. The method of claim 69, wherein the first primer comprises a poly-T portion.

75. The method of claim 1, wherein the first primer comprises a 5′

portion that is not hybridizable to the target RNA.

2. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising the steps of:

(a) extending a first primer hybridized to a target RNA with an RNA-dependent DNA polymerase, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) cleaving RNA in the complex of step (a) with an enzyme that cleaves RNA from an RNA/DNA hybrid;

(c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase, whereby a complex comprising the first primer extension product and a second primer extension product is produced;

(d) denaturing the complex of step (c);

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

(f) extending an exponential amplification primer hybridized to said RNA transcripts with an RNA-dependent DNA polymerase, whereby a complex comprising an exponential amplification primer extension product and an RNA transcript is produced;

(g) cleaving RNA in the complex of step (f) with an enzyme that cleaves RNA from an RNA/DNA hybrid;

(h) hybridizing a second propromoter polynucleotide comprising a propromoter and a region which hybridizes to a single stranded exponential amplification primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the target RNA;

(i) optionally repeating steps (f) to (h);

whereby multiple copies of the complementary sequence of the RNA sequence of interest are produced.

12. The method of claim 2, wherein the first primer comprises a 5′

portion that is not hybridizable to the target RNA.

13. The method of claim 12, wherein said 5′

portion comprises a sequence the complement of which is hybridizable by the propromoter polynucleotide in step (e) or (h).

16. The method of claim 2, wherein the first primer comprises a 5′

portion that is not hybridizable to the target RNA, and wherein said 5′

portion comprises a sequence the complement of which is hybridizable by the propromoter polynucleotide in step (e) or (h).

17. The method of claim 2, wherein the second primer and the exponential amplification primer are the same.

18. The method of claim 2, wherein the second primer and the exponential amplification primer are different.

19. The method of claim 18, wherein the second primer and the exponential amplification primer hybridize to different complementary sequences.

39. The method of any of claims 2, 7, and 9, wherein the propromoter polynucleotide comprises a region at the 3′

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

40. The method of claim 39, wherein the propromoter polynucleotide is a propromoter template oligonucleotide.

6. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a target RNA;

(b) a first primer comprising a sequence that is hybridizable to the target RNA;

(c) a second primer comprising a sequence hybridizable to an extension product of the first primer;

(d) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded second primer extension product;

(e) an RNA-dependent DNA polymerase;

(f) a DNA-dependent DNA polymerase;

(g) an RNA polymerase; and

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

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, propromoter polynucleotide hybridization, and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

26. The method of any of claims 6, 7, 8, and 9, wherein the first primer comprises a 5′

sequence that is not hybridizable to the target RNA.

27. The method of claim 26, wherein the 5′

sequence comprises a sequence the complement of which is hybridizable by a propromoter polynucleotide.

28. The method of any of claims 6, 7, 8, and 9, wherein the first primer comprises a poly-T sequence;

and the target RNA is mRNA.

41. The method of any of claims 6, 7, 8, and 9, wherein at least one type of rNTP used is a labeled rNTP, whereby labeled products are generated.

60. A method of characterizing an RNA sequence of interest, comprising analyzing labeled RNA products, said labeled RNA products produced by amplifying a target RNA by the method of claim 41.

61. The method of claim 60, wherein analyzing comprises contacting the labeled DNA products with at least one probe.

62. The method of claim 61, wherein the at least one probe is provided as a microarray.

63. The method of claim 62, wherein the microarray comprises at least one 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.

64. The method of claim 63, wherein the probe is immobilized on the substrate in a two-dimensional configuration or a three-dimensional configuration comprising pins, rods, fibers, tapes, threads, beads, particles, microtiter wells, capillaries, and cylinders.

65. The method of claim 60, wherein the target RNA is mRNA.

66. The method of claim 60, wherein analyzing the labeled RNA products comprises determining amount of said products, whereby the amount of the RNA sequence of interest present in a sample is quantified.

7. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a target RNA;

(b) a first primer comprising a sequence that is hybridizable to the target RNA;

(c) a second primer comprising a sequence hybridizable to an extension product of the first primer;

(d) an exponential amplification primer comprising a sequence hybridizable to an RNA transcript comprising a sequence complementary to the target RNA;

(e) a first propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded second primer extension product;

(f) a second propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded exponential amplification primer extension product;

(g) an RNA-dependent DNA polymerase;

(h) a DNA-dependent DNA polymerase;

(i) an RNA polymerase; and

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

wherein the incubation is conditions that permit primer hybridization, primer extension, RNA cleavage, propromoter polynucleotide hybridization, and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

29. The method of claim 7, wherein the second primer and the exponential amplification primer are the same.

30. The method of claim 7, wherein the second primer and the exponential amplification primer are different.

31. The method of claim 30, wherein the second primer and the exponential amplification primer hybridize to different complementary sequences.

8. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a target RNA;

(b) a first primer comprising a sequence that is hybridizable to the target RNA;

(c) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded second strand cDNA, said second strand cDNA complementary to an extension product of the first primer;

(d) an RNA-dependent DNA polymerase;

(e) a DNA-dependent DNA polymerase;

(f) an RNA polymerase; and

(g) an enzyme that cleaves RNA from an RNA/DNA hybrid,

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, propromoter polynucleotide hybridization, and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

9. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a target RNA;

(b) a first primer comprising a sequence that is hybridizable to the target RNA;

(c) an exponential amplification primer comprising a sequence hybridizable to an RNA transcript comprising a sequence complementary to the target RNA;

(d) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded second strand cDNA, said second strand cDNA complementary to an extension product of the first primer;

(e) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded exponential amplification primer extension product;

(f) an RNA-dependent DNA polymerase;

(g) a DNA-dependent DNA polymerase;

(h) an RNA polymerase; and

(i) an enzyme that cleaves RNA from an RNA/DNA hybrid,

wherein the incubation is conditions that permit primer hybridization, primer extension, RNA cleavage, propromoter polynucleotide hybridization, and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

10. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising the steps of:

(a) synthesizing a first primer extension product by extending a first primer hybridized to a target RNA, whereby a complex comprising the first primer extension product and the target RNA is produced;

(b) synthesizing a second primer extension product by extending a second primer hybridized to the first primer extension product, whereby a complex comprising the first primer extension product and the second primer extension product is produced;

(c) denaturing the complex of step (b);

(d) hybridizing to the second primer extension product a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the second primer extension product; and

(e) synthesizing RNA transcripts comprising sequences complementary to the target RNA,

whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.

76. The method of claim 10 or 11, wherein the first primer comprises a 5′

portion that is not hybridizable to the target RNA.

77. The method of claim 76, wherein said 5′

portion comprises a sequence the complement of which is hybridizable to one or more of the propromoter polynucleotides of steps (d) and (g).

78. The method of claim 10 or 11, wherein the target RNA is mRNA.

84. The method of claim 10 or 11, wherein the synthesizing of RNA transcripts is carried out in the presence of at least one type of labeled rNTP, whereby labeled products are generated.

85. The method of claim 10 or 11, wherein the first primer is a random primer.

86. The method of claim 10 or 11, wherein the second primer comprises DNA.

87. The method of claim 10 or 11, wherein the second primer comprises a fragment of the target RNA hybridized to the primer extension product, said fragment generated by cleaving RNA in the complex of step (a).

88. The method of claim 10 or 11, wherein said method comprises generating multiple copies of two or more different RNA sequences of interest.

89. The method of claim 88, wherein said method comprises at least two different first primers.

90. The method of claim 85, wherein the first and second propromoter polynucleotides are the same.

91. The method of claim 85, wherein the first and second propromoter polynucleotides are different.

11. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising the steps of:

(a) synthesizing a first primer extension product by extending a first primer hybridized to a target RNA, whereby a complex comprising the first primer extension product and the target RNA is produced;

(b) synthesizing a second primer extension product by extending a second primer hybridized to the first primer extension product, whereby a complex comprising the first primer extension product and the second primer extension product is produced;

(c) denaturing the complex of step (b);

(d) hybridizing to the second primer extension product a first propromoter polynucleotide comprising a propromoter and a region which hybridizes to the second primer extension product;

(e) synthesizing RNA transcripts comprising sequences complementary to the target RNA, such that RNA transcripts are produced comprising sequences complementary to the target RNA;

(f) synthesizing a single stranded exponential amplification primer extension product by extending an exponential amplification primer hybridized to said RNA transcripts, whereby a complex comprising an exponential amplification primer extension product and an RNA transcript is produced;

(g) synthesizing RNA transcripts comprising sequences complementary to the target RNA by hybridizing a second propromoter polynucleotide comprising a propromoter and a region which hybridizes to the single stranded exponential amplification primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the target RNA; and

(h) optionally repeating steps (f) to (g);

whereby multiple copies of the complementary sequence of the RNA sequence of interest are produced.

44. A method of sequencing an RNA sequence of interest, said method comprising analyzing amplification products to determine sequence, said amplification products produced by the method of any of claims 2, 7, 9, and 11, wherein RNA transcripts generated from the second primer extension product are amplified in the presence of a mixture of rNTPs and rNTP analogs such that transcription is terminated upon incorporation of an rNTP analog.

45. The method of claim 44, wherein the target RNA is mRNA.

80. The method of claim 11, wherein the first primer comprises a 5′

portion that is not hybridizable to the target RNA, and wherein said 5′

portion comprises a sequence the complement of which is hybridizable to one or more of the propromoter polynucleotides of steps (d) and (g).

81. The method of claim 11, wherein the second primer and the exponential amplification primer are the same.

82. The method of claim 11, wherein the second primer and the exponential amplification primer are different.

83. The method of claim 82, wherein the second primer and the exponential amplification primer hybridize to different complementary sequences.

72. A method for generating multiple copies of a sequence complementary to an RNA sequence of interest comprising:

(a) hybridizing to a single stranded second primer extension product, a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the second primer extension product; and

(b) synthesizing RNA transcripts comprising sequences complementary to the target RNA whereby multiple copies of the sequence complementary to the RNA sequence of interest are generated,

wherein the second primer extension product is prepared by synthesizing a first primer extension product by extending a first primer hybridized to the RNA sequence of interest, whereby a first complex comprising the first primer extension product and the RNA sequence of interest is produced, then synthesizing a second primer extension product by extending a second primer hybridized to the first primer extension product, whereby a second complex comprising the first primer extension product and the second primer extension product is produced, and denaturing the second complex.

73. A method of performing subtractive hybridization, said method comprising:

hybridizing multiple DNA copies of the complement of one or more RNA sequences of interest from a first RNA population to a second mRNA population, said multiple DNA copies prepared using the method of claim 72 whereby a subpopulation of the second mRNA population forms a complex with the DNA copies.

74. A method of performing subtractive hybridization, said method comprising:

(a) hybridizing multiple DNA copies of the complement of at least two RNA sequences of interest to a second mRNA population, said multiple DNA copies prepared from a first RNA population using the method of claim 72;

whereby a subpopulation of the second mRNA population forms a complex with a DNA copy;

(b) cleaving RNA in the complex of step (a) with an enzyme that cleaves RNA from an RNA/DNA hybrid; and

(c) amplifying the unhybridized subpopulation of the second mRNA population, whereby multiple copies of single stranded DNA complementary to the unhybridized subpopulation of the second mRNA population are generated.

79. The method of claim 72, wherein the first primer comprises a poly-dT sequence.

92. The method of claim 72, wherein the first primer comprises a 5′

portion that is not hybridizable to the RNA sequence of interest.

93. The method of claim 72, wherein the RNA sequence of interest is mRNA.

94. The method of claim 93, wherein the first primer comprises a poly-dT sequence.

95. The method of claim 72, wherein the synthesis of RNA transcripts is carried out in the presence of at least one type of labeled rNTP, whereby labeled products are generated.

96. The method of claim 72, wherein the first primer is a random primer.

97. The method of claim 72, wherein said method comprises generating multiple copies of two or more different RNA sequences of interest.

98. The method of claim 97, wherein said method comprises at least two different first primers.