IMPROVED NUCLEIC ACID AMPLIFICATION PROCEDURE

• US 20090130721A1
• Filed: 09/07/2006
• Published: 05/21/2009
• Est. Priority Date: 09/07/2005
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith, Wang, Shenglong
• Granted Patent: US 7,939,258 B2
• Time in Patent Office: Days
• US Class Current: 435/91.210
• 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]

Abstract

The invention provides methods for amplification of polynucleotide sequences using primers containing single-stranded RNA. The methods employ use of an enzyme capable of cleaving single-stranded RNA, such as RNase I, to degrade a first RNA-containing primer prior to addition of a second RNA-containing primer. 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 a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising the steps of:

(a) extending a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) cleaving the target RNA in the complex of step (a);

(c) extending a second primer hybridized to the first primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity and at least one enzyme comprising RNA-dependent DNA polymerase activity, whereby a second primer extension product is produced to form a complex of first and second primer extension products;

(d) incubating the reaction mixture comprising the products of step (c) and excess first primer, if any, with an enzyme that is capable of cleaving single-stranded RNA;

(e) inactivating the enzyme that is capable of cleaving single-stranded RNA;

(f) cleaving RNA from the first primer in the complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite amplification primer hybridizes to the second primer extension product, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion; and

(g) extending the composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity;

whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, and whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

5. The method of any of claim 1, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

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

RNA portion of the first primer is adjacent to the 3′

DNA portion.

7. The method of claim 1, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions in which the first primer hybridizes to the target RNA.

8. The method of claim 1, wherein the first primer comprises a random primer sequence.

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

DNA portion of the first primer comprises a poly-dT sequence.

10. The method of claim 9, wherein the 3′

DNA portion of the first primer comprises at least 1 random nucleotide at the 3′

end.

11. The method of claim 1, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

12. The method of claim 11, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

13. The method of claim 1, wherein the target RNA is mRNA.

14. The method of claim 1, wherein the second primer comprises a fragment of the target RNA hybridized to the first primer extension product.

15. The method claim 1, wherein the second primer comprises DNA.

16. The method of claim 1, wherein the second primer is a random primer.

17. The method of claim 1, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

18. The method of claim 1, wherein the enzyme capable of cleaving single-stranded RNA is RNase I.

19. The method of claim 18, wherein said inactivating said RNase I comprises inactivation by heat.

20. The method of claim 1, wherein step (g) comprises extension of said composite amplification primer in the presence of a non-canonical nucleotide.

23. The method of any of claims 20 to 22, wherein the non-canonical nucleotide is dUTP.

24. The method of claim 1, wherein step (g) comprises extension of said composite amplification primer in the presence of a labeled nucleotide.

25. The method of claim 1, wherein step (g) comprises extension of said composite amplification primer in the presence of a labeled nucleotide terminator.

30. The method of any of claims 1, 3, or 4, wherein said multiple copies of a polynucleotide sequence complementary to a sequence of interest are contacted with an enzyme that is capable of cleaving single-stranded RNA, wherein RNA from the composite amplification primer is cleaved.

31. The method of claim 30, wherein the enzyme that is capable of cleaving single-stranded RNA is RNase I.

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

(a) extending a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) cleaving the target RNA in the complex of step (a);

(c) extending a second primer hybridized to the first primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity and at least one enzyme comprising RNA-dependent DNA polymerase activity, whereby a second primer extension product is produced to form a complex of first and second primer extension products;

(d) incubating the reaction mixture comprising the products of step (c) and excess first primer, if any, with an enzyme that is capable of cleaving single-stranded RNA;

(e) inactivating the enzyme that is capable of cleaving single-stranded RNA;

(f) cleaving RNA from the first primer in the complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite amplification primer hybridizes to the second primer extension product, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion;

(g) extending said composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity, whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated; and

(h) hybridizing the displaced first primer extension product with a polynucleotide comprising a propromoter and a region which is hybridizable to the displaced first 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 first primer extension product,

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

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

(a) incubating a reaction mixture, said reaction mixture comprising;

(i) a target RNA;

(ii) a first primer that is hybridizable to a target RNA, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion; and

(iii) at least one enzyme comprising RNA-dependent DNA polymerase activity wherein the incubation is under conditions that permit primer hybridization and primer extension, whereby a complex comprising a first primer extension product and the target RNA is formed;

(b) incubating a reaction mixture, said reaction mixture comprising;

(i) the first primer extension product;

(ii) a second primer;

(iii) at least one enzyme comprising DNA-dependent DNA polymerase activity;

(iv) at least one enzyme comprising RNA-dependent DNA polymerase activity; and

(v) optionally, at least one enzyme capable of cleaving RNA from an RNA/DNA hybrid;

wherein the incubation is under conditions permitting formation of a complex comprising the first primer extension product and a second primer extension product;

(c) incubating at least a portion of the reaction mixture of (b) with an enzyme capable of cleaving single-stranded RNA;

(d) inactivating the enzyme capable of cleaving single-stranded RNA; and

(e) incubating a reaction mixture, said reaction mixture comprising;

(i) the reaction products generated according to steps (a), (b), (c), and (d);

(ii) at least one enzyme capable of cleaving RNA from an RNA/DNA hybrid;

(iii) a composite amplification primer, wherein the composite amplification primer comprises a RNA portion and a 3′

DNA portion;

(iv) at least one enzyme comprising DNA-dependent DNA polymerase activity, wherein the incubation is under conditions that permit cleavage of RNA, composite primer hybridization, primer extension, and displacement of the first primer extension product from the complex comprising the first primer extension product and the second primer extension product, whereby another composite amplification primer hybridizes and primer extension and strand displacement are repeated;

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

21. The method of claim 3, wherein the reaction mixture of step (e) further comprises a non-canonical nucleotide.

26. The method of claim 3, wherein the reaction mixture of step (e) further comprises a labeled nucleotide.

27. The method of claim 3, wherein the reaction mixture of step (e) further comprise a labeled nucleotide terminator.

4. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising:

(i) contacting a reaction mixture comprising a complex of first and second primer extension products and a first primer with an enzyme capable of cleaving single-stranded RNA, wherein the first primer extension product is produced by extension of the first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, wherein the second primer extension product is generated by extension of a second primer hybridized to the first primer extension product;

(ii) inactivating the enzyme capable of cleaving single-stranded RNA; and

(iii) contacting the complex of first and second primer extension products with a composite amplification primer and at least one enzyme that cleaves RNA from an RNA/DNA hybrid, said composite amplification primer comprising an RNA portion and a 3′

DNA portion, wherein the composite amplification primer hybridizes to the second primer extension product;

whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated; and

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

22. The method of claim 4, wherein step (iii) comprises extension of said composite amplification primer in the presence of a non-canonical nucleotide.

28. The method of claim 4, wherein step (iii) comprises extension of said composite amplification primer in the presence of a labeled nucleotide.

29. The method of claim 4, wherein step (iii) comprises extension of composite amplification primer in the presence of a labeled nucleotide terminator.

32. A method for amplification of a polynucleotide template, comprising:

(a) extending a first primer hybridized to the polynucleotide template with at least one enzyme comprising a DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, whereby a complex comprising a first primer extension product and the template polynucleotide is produced;

(b) extending a second primer hybridized to the first primer extension product to produce a complex comprising a first primer extension product and a second primer extension product;

(c) incubating the reaction mixture comprising the products of step (b) and excess first primer, if any, with an enzyme that is capable of cleaving single-stranded R A, whereby unhybridized single-stranded RNA of the RNA portion of the first primer is cleaved;

(d) inactivating the enzyme that is capable of cleaving single-stranded RNA;

(e) cleaving RNA from the first primer in the complex of first primer extension product and second primer extension product with at least one enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite amplification primer hybridizes to the second primer extension product, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion; and

(f) extending the composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity, whereby the first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, and whereby multiple copies of a polynucleotide amplification product are generated.

35. The method of claim 32, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

36. The method of claim 35, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion.

37. The method of claim 32, wherein the first primer is hybridizable to a multiplicity of template polynucleotide sites.

38. The method of claim 32, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

39. The method of claim 38, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

40. The method of claim 32, wherein the template polynucleotide is DNA.

41. The method of claim 32, wherein the template polynucleotide is RNA.

42. The method of claim 32, wherein the first primer is at least two different primers.

43. The method of claim 32, wherein the first primer comprises a random sequence.

44. The method of claim 32, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

45. The method of claim 32, wherein the enzyme capable of cleaving single-stranded RNA is RNase I.

46. The method of claim 45, wherein said inactivating said RNase I comprises inactivation by heat.

47. The method of claim 32, wherein step (f) comprises extension of said composite amplification primer in the presence of a non-canonical nucleotide.

50. The method of any of claims 47 to 49, wherein the non-canonical nucleotide is dUTP.

51. The method of claim 32, wherein step (f) comprises extension of said composite amplification primer in the presence of a labeled nucleotide.

52. The method of claim 32, wherein step (f) comprises extension of said composite amplification primer in the presence of a labeled nucleotide terminator.

57. The method of any of claims 32, 33 or 34, wherein said multiple copies of a polynucleotide amplification product are contacted with an enzyme that is capable of cleaving single-stranded RNA, wherein RNA from the composite amplification primer is cleaved.

58. The method of claim 57, wherein the enzyme that is capable of cleaving single-stranded RNA is RNase I.

33. A method for amplification of a template polynucleotide, comprising:

(a) incubating a reaction mixture, said reaction mixture comprising;

(i) a template polynucleotide;

(ii) a first primer, wherein the first primer is a composite primer that is hybridizable to at least one template site, wherein the composite primer comprises an RNA portion and a 3′

DNA portion; and

(iii) a DNA polymerase;

wherein the incubation is under conditions that permit primer hybridization and primer extension, whereby a complex comprising a first primer extension product and the template polynucleotide is formed;

(b) incubating a reaction mixture, said reaction mixture comprising;

(i) the first primer extension product;

(ii) a second primer;

(iii) at least one enzyme comprising DNA-dependent DNA polymerase activity;

(iv) at least one enzyme comprising RNA-dependent DNA polymerase activity; and

(v) optionally, at least one enzyme capable of cleaving RNA from an RNA/DNA hybrid;

wherein the incubation is under conditions permitting formation of a complex comprising the first primer extension product and a second primer extension product;

(c) incubating at least a portion of the reaction mixture of (b) with an enzyme capable of cleaving single-stranded RNA;

(d) inactivating the enzyme capable of cleaving single-stranded RNA; and

(e) incubating a reaction mixture, said reaction mixture comprising;

(i) the reaction products generated according to steps (a), (b), (c), and (d);

(ii) at least one enzyme capable of cleaving RNA from an RNA/DNA hybrid;

(iii) a composite amplification primer, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion;

(iv) at least one enzyme comprising DNA-dependent DNA polymerase activity, wherein the incubation is Linder conditions that permit cleavage of RNA, composite primer hybridization, primer extension, and displacement of the first primer extension product from the complex comprising the first primer extension product and the second primer extension product, whereby another composite amplification primer hybridizes and primer extension and strand displacement are repeated;

whereby multiple copies of a polynucleotide amplification product are generated.

48. The method of claim 33, wherein the reaction mixture of step (e) further comprises a non-canonical nucleotide.

53. The method of claim 33, wherein the reaction mixture of step (e) further comprises a labeled nucleotide.

54. The method of claim 33, wherein the reaction mixture of step (e) further comprises a labeled nucleotide terminator.

34. A method for amplification of a template polynucleotide, said method comprising:

(i) contacting a reaction mixture comprising a first primer and a complex of first primer extension product and second primer extension product with an enzyme capable of cleaving single-stranded RNA, wherein the complex of first primer extension product and second primer extension product is produced by extension of a first primer hybridized to the template polynucleotide with a DNA polymerase, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, wherein the second primer extension product is generated by extension of a second primer hybridized to the first primer extension product;

(ii) inactivating the enzyme capable of cleaving single-stranded RNA; and

(iii) contacting the complex of first primer extension product and second primer extension product with at least one enzyme that cleaves RNA from an RNA/DNA hybrid and a composite amplification primer, said composite amplification primer comprising an RNA portion and a 3′

DNA portion, wherein the composite amplification primer hybridizes to the second primer extension product;

whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer, and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide amplification product are generated.

49. The method of claim 34, wherein step (iii) comprises extension of said composite amplification primer in the presence of a non-canonical nucleotide.

55. The method of claim 34, wherein step (iii) comprises extension of said composite amplification primer in the presence of a labeled nucleotide.

56. The method of claim 34, wherein step (iii) comprises extension of said composite amplification primer in the presence of a labeled nucleotide terminator.

59. 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 polynucleotide template comprising the target sequence; and

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

DNA portion, wherein the composite primer is hybridized to the polynucleotide 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 and strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced; and

(c) incubating said multiple copies of the complementary sequence of the target sequence with an enzyme that is capable of cleaving single-stranded RNA, wherein RNA from the composite primer is cleaved.

61. The method of claim 59 or 60, wherein the enzyme capable of cleaving single-stranded RNA is RNase I.

60. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising:

(a) extending a composite amplification primer in a complex comprising;

(i) a complex of first and second primer extension products, wherein the first primer extension product is produced by extension of a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, wherein the second primer extension product is generated by extension of a second primer hybridized to the first primer extension product, and wherein RNA from the complex of first and second primer extension products is cleaved with at least one enzyme that cleaves RNA from an RNA/DNA hybrid; and

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

DNA portion, wherein the composite amplification primer is hybridized to the second primer extension product;

whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated; and

(b) contacting said multiple copies of the complementary sequence of the RNA sequence of interest with an enzyme that is capable of cleaving single-stranded RNA, wherein RNA from the composite primer is cleaved.

62. A kit comprising a first composite primer, a second composite primer, and an enzyme capable of cleaving single-stranded RNA, wherein the first composite primer comprises an RNA portion and a 3′

DNA portion, wherein the second composite primer comprises an RNA portion and a 3′

DNA portion, and wherein the second composite primer comprises a sequence that is hybridizable to a polynucleotide comprising a complement of the first composite primer.

63. The kit of claim 62, wherein the RNA portion of the first composite primer is 5′

with respect to the 3′

DNA portion of the first composite primer and the RNA portion of the second composite primer is 5′

with respect to the 3′

DNA portion of the second composite primer.

64. The kit of claim 63, wherein the 5′

portion of the first composite primer is adjacent to the 3′

DNA portion of the first composite primer and the 5′

RNA portion of the second composite primer is adjacent to the 3′

DNA portion of the second composite primer.

65. The kit of claim 62, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to a target RNA under conditions wherein the first composite primer hybridizes to the target RNA.

66. The kit of claim 62, wherein the 3′

DNA portion of the first composite primer comprises a poly-dT sequence.

67. The kit of claim 66, wherein the 3′

DNA portion of the first composite primer comprises at least 1 random nucleotide at the 3′

end.

68. The kit of claim 62, wherein the enzyme capable of cleaving single-stranded RNA is RNase I.

69. The kit of claim 62, further comprising an enzyme that cleaves RNA from an RNA/DNA hybrid.

70. The kit of claim 69, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNaseH.

71. The kit of claim 62, further comprising an RNA-dependent DNA polymerase.

72. The kit of claim 62, further comprising a non-canonical nucleotide.

73. The kit of claim 72, wherein the non-canonical nucleotide is dUTP.

74. The kit of claim 62, further comprising a labeled nucleotide.

75. The kit of claim 62, further comprising a labeled nucleotide terminator.