Kits for Performing Amplification Reactions

• US 20070299254A1
• Filed: 08/26/2005
• Published: 12/27/2007
• Est. Priority Date: 08/27/2004
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Lam, Wal-Chung, Schroder, Astrid, Kolk, Daniel, Brentano, Steven, Livezey, Kristin, Becker, Michael
• Granted Patent: US 7,696,337 B2
• Time in Patent Office: Days
• US Class Current: 536/24.33
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• 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 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/163 blocking probe

Abstract

The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side products. The method uses only one primer, the “priming oligonucleotide,” a 3'"'"'blocked promoter oligonucleotide and optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or eliminating the formation of side products. The method of the present invention minimizes or eliminates the emergence of side products, thus providing a high level of specificity. Furthermore, the appearance of side products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or eliminates this problem, thus providing an enhanced level of sensitivity.

Claims

1-13. -13. (canceled)

14. A kit for use in synthesizing multiple copies of an RNA target sequence contained within a target nucleic acid, said kit comprising:

a priming oligonucleotide which hybridizes to the 3′

-end of said RNA target sequence and primes the synthesis of a first DNA primer extension product complementary to said RNA target sequence, wherein said priming oligonucleotide is substantially comprised of deoxynucleotides;

a promoter oligonucleotide comprising a first region which hybridizes to a 3′

-region of said first DNA primer extension product and a second region which is a promoter for an RNA polymerase, said promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom; and

a binding molecule which binds to said target nucleic acid adjacent to or near the 5′

-end of said RNA target sequence.

15. The kit of claim 14, wherein said priming oligonucleotide consists of deoxynucleotides and/or analogs thereof.

16. The kit of claim 14, wherein a 5′

-region of said priming oligonucleotide includes one or more modifications for increasing the binding affinity of said priming oligonucleotide for said RNA target sequence, and wherein said modifications do not prevent said priming oligonucleotide from being extended in a primer extension reaction.

17. The kit of claim 14 further comprising a cap which hybridizes to a region at the 3′

-end of said priming oligonucleotide, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom.

18. The kit of claim 14, wherein said kit includes only a single priming oligonucleotide.

19. The kit of claim 14, wherein said promoter oligonucleotide is modified to include a blocking moiety at its 3′

-terminus.

20. The kit of claim 19, wherein said blocking moiety of said promoter oligonucleotide comprises a substituent selected from the group consisting of:

a modified nucleotide, a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation, a 3′

alkyl group, a 3′

2′

-dideoxynucleotide, a 3′

cordycepin, a 3′

alkane-diol residue, a 3′

non-nucleotide moiety, a nucleic acid binding protein, and mixtures thereof.

21. The kit of claim 14, wherein said promoter oligonucleotide further comprises an insertion sequence positioned between or adjacent to said first and second regions, and wherein the presence of said insertion sequence in said promoter oligonucleotide enhances the rate at which copies of said RNA target sequence are formed.

22. The kit of claim 19, wherein said first region of said promoter oligonucleotide is hybridized to an oligodeoxynucleotide, and wherein said first region comprises a 3′

-region having a sufficient number of contiguous ribonucleotides hybridized to said oligodeoxynucleotide such that said blocking moiety of said promoter oligonucleotide is released in the presence of an enzymatic activity capable of cleaving said region of ribonucleotides.

23. The kit of claim 22, wherein said promoter oligonucleotide comprises said oligodeoxynucleotide.

24. The kit of claim 14, wherein any oligonucleotide provided in said kit which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom.

25. The kit of claim 14, wherein said binding molecule is a terminating oligonucleotide.

26. The kit of claim 25, wherein said terminating oligonucleotide comprises a blocking moiety situated at its 3′

-terminus to prevent the initiation of DNA synthesis therefrom.

27. The kit of claim 14, wherein said binding molecule is a modifying oligonucleotide.

28. The kit of claim 14 further comprising an extender oligonucleotide which hybridizes to said first DNA primer extension product 3′

to said promoter oligonucleotide.

29. The kit of claim 28, wherein said extender oligonucleotide comprises a blocking moiety situated at its 3′

terminus to prevent the initiation of DNA synthesis therefrom.

30. The kit of claim 14 further comprising a reverse transcriptase.

31. The kit of claim 30, wherein said reverse transcriptase has an RNase H activity.

32. The kit of claim 30 further comprising an enzyme other than said reverse transcriptase which has an RNase H activity.

33. The kit of claim 14 further comprising a detection probe which hybridizes to said RNA target sequence or its complement to give a detectable signal during synthesis.

34. A kit for use in synthesizing multiple copies of a DNA target sequence contained within a target nucleic acid, said kit comprising:

a promoter oligonucleotide comprising a first region which hybridizes to a 3′

-region of said DNA target sequence present in a target nucleic acid to form a promoter oligonucleotide;

target nucleic acid hybrid and a second region which is a promoter for an RNA polymerase, said promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom;

a priming oligonucleotide which hybridizes to the 3′

-end of an RNA product transcribed from said promoter oligonucleotide;

target nucleic acid hybrid and primes the synthesis of a first DNA primer extension product complementary to said RNA product, wherein said RNA product comprises a base region complementary to said DNA target sequence,

provided that said kit does not include a priming oligonucleotide which hybridizes to said target nucleic acid and primes the synthesis of a primer extension product complementary to said DNA target sequence, and

further provided that said kit does not include a restriction endonuclease capable of cleaving a double-stranded complex comprising said target nucleic acid.

35. The kit of claim 34, wherein said promoter oligonucleotide is modified to include a blocking moiety at its 3′

-terminus.

36. The kit of claim 34, wherein said blocking moiety of said promoter oligonucleotide comprises a substituent selected from the group consisting of:

a modified nucleotide, a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation, a 3′

alkyl group, a 3′

2′

-dideoxynucleotide, a 3′

cordycepin, a 3′

alkane-diol residue, a 3′

non-nucleotide moiety, a nucleic acid binding protein, and mixtures thereof.

37. The kit of claim 34, wherein said promoter oligonucleotide further comprises an insertion sequence positioned between or adjacent to said first and second regions, and wherein the presence of said insertion sequence in said promoter oligonucleotide enhances the rate at which said RNA products are formed.

38. The kit of claim 35, wherein said first region of said promoter oligonucleotide is hybridized to an oligodeoxynucleotide, and wherein said first region comprises a 3′

-region having a sufficient number of contiguous ribonucleotides hybridized to said oligodeoxynucleotide such that said blocking moiety of said promoter oligonucleotide is released in the presence of an enzymatic activity capable of cleaving said region of ribonucleotides.

39. The kit of claim 38, wherein said promoter oligonucleotide comprises said oligodeoxynucleotide.

40. The kit of claim 34, wherein any oligonucleotide provided in said kit which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom.

41. The kit of claim 34, wherein said priming oligonucleotide consists of deoxynucleotides and/or analogs thereof.

42. The kit of claim 34, wherein a 5′

-region of said priming oligonucleotide includes one or more modifications for increasing the binding affinity of said priming oligonucleotide for said RNA product, and wherein said modifications do not prevent said priming oligonucleotide from being extended in a primer extension reaction.

43. The kit of claim 34 further comprising a cap which hybridizes to a region at the 3′

-end of said priming oligonucleotide, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom.

44. The kit of claim 34 further comprising an extender oligonucleotide which hybridizes to said DNA target sequence or to said first DNA primer extension product 3′

to said promoter oligonucleotide.

45. The kit of claim 44, wherein said extender oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom.

46. The kit of claim 34 further comprising a reverse transcriptase.

47. The kit of claim 46, wherein said reverse transcriptase has an RNase H activity.

48. The kit of claim 46 further comprising an enzyme other than said reverse transcriptase which has an RNase H activity.

49. The kit of claim 34 further comprising a detection probe which hybridizes to said DNA target sequence or its complement to give a detectable signal during synthesis.

50. A kit for use in synthesizing multiple copies of an RNA target sequence, said kit comprising:

a priming oligonucleotide which hybridizes to the 3′

-end of said RNA target sequence and primes the synthesis of a first DNA primer extension product complementary to said RNA target sequence;

a promoter oligonucleotide comprising a first region which hybridizes to a 3′

-region of said first DNA primer extension product and a second region which is a promoter for an RNA polymerase, said promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom; and

an extender oligonucleotide which hybridizes to said first DNA primer extension product 3′

to said promoter oligonucleotide.

51. The kit of claim 50, wherein said priming oligonucleotide consists of deoxynucleotides and/or analogs thereof.

52. The kit of claim 50, wherein a 5′

-region of said priming oligonucleotide includes one or more modifications for increasing the binding affinity of said priming oligonucleotide for said RNA target sequence, and wherein said modifications do not prevent said priming oligonucleotide from being extended in a primer extension reaction.

53. The kit of claim 50 further comprising a cap which hybridizes to a region at the 3′

-end of said priming oligonucleotide, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom.

54. The kit of claim 50, wherein said promoter oligonucleotide is modified to include a blocking moiety at its 3′

-terminus.

55. The kit of claim 54, wherein said blocking moiety of said promoter oligonucleotide comprises a substituent selected from the group consisting of:

a modified nucleotide, a nucleotide or a nucleotide sequence having a 3′

-to-5′

orientation, a 3′

alkyl group, a 3′

2′

-dideoxynucleotide, a 3′

cordycepin, a 3′

alkane-diol residue, a 3′

non-nucleotide moiety, a nucleic acid binding protein, and mixtures thereof.

56. The kit of claim 50, wherein said promoter oligonucleotide further comprises an insertion sequence positioned between or adjacent to said first and second regions, and wherein the presence of said insertion sequence in said promoter oligonucleotide enhances the rate at which copies of said RNA target sequence are formed.

57. The kit of claim 54, wherein said first region of said promoter oligonucleotide is hybridized to an oligodeoxynucleotide, and wherein said first region comprises a 3′

-region having a sufficient number of contiguous ribonucleotides hybridized to said oligodeoxynucleotide such that said blocking moiety of said promoter oligonucleotide is released in the presence of an enzymatic activity capable of cleaving said region of ribonucleotides.

58. The kit of claim 57, wherein said promoter oligonucleotide comprises said oligodeoxynucleotide.

59. The kit of claim 50, wherein any oligonucleotide provided in said kit which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom.

60. The kit of claim 50, wherein said extender oligonucleotide comprises a blocking moiety situated at its 3′

terminus to prevent the initiation of DNA synthesis therefrom.

61. The kit of claim 50 further comprising a reverse transcriptase.

62. The kit of claim 61, wherein said reverse transcriptase has an RNase H activity.

63. The kit of claim 61 further comprising an enzyme other than said reverse transcriptase which has an RNase H activity.

64. The kit of claim 50 further comprising a detection probe which hybridizes to said RNA target sequence or its complement to give a detectable signal during synthesis.

65. A kit for use in synthesizing multiple copies of an RNA target sequence, said kit comprising:

a priming oligonucleotide which hybridizes to the 3′

-end of said RNA target sequence and primes the synthesis of a first DNA primer extension product complementary to said RNA target sequence; and

a cap which hybridizes to a region at the 3′

-end of said priming oligonucleotide, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom.

66. The kit of claim 65, wherein said priming oligonucleotide consists of deoxynucleotides and/or analogs thereof.

67. The kit of claim 65, wherein a 5′

-region of said priming oligonucleotide includes one or more modifications for increasing the binding affinity of said priming oligonucleotide for said RNA target sequence, and wherein said modifications do not prevent said priming oligonucleotide from being extended in a primer extension reaction.

68. The kit of claim 65, wherein said cap prevents non-specific hybridization between said priming oligonucleotide and any other oligonucleotide of said kit when said cap is hybridized to said priming oligonucleotide.

69. The kit of claim 65, wherein each said cap is a capping oligonucleotide modified to include a blocking moiety situated at its 3′

-terminus.

70. The kit of claim 65, wherein the 3′

-end of said cap is covalently attached to the 5′

-end of said priming oligonucleotide, and wherein said cap hybridizes to the 3′

-end of said priming oligonucleotide by forming a loop.

71. The kit of claim 65 further comprising a promoter oligonucleotide comprising a first region which hybridizes to a 3′

-region of said first DNA primer extension product and a second region which is a promoter for an RNA polymerase, said promoter oligonucleotide being modified to prevent the initiation of DNA synthesis therefrom.

72. The kit of claim 71, wherein said promoter oligonucleotide further comprises an insertion sequence positioned between or adjacent to said first and second regions, and wherein the presence of said insertion sequence in said promoter oligonucleotide enhances the rate at which copies of said RNA target sequence are formed.

73. The kit of claim 71, wherein said promoter oligonucleotide is modified to include a blocking moiety at its 3′

-terminus, wherein said first region of said promoter oligonucleotide is hybridized to an oligodeoxynucleotide, and wherein said first region comprises a 3′

-region having a sufficient number of contiguous ribonucleotides hybridized to said oligodeoxynucleotide such that said blocking moiety of said promoter oligonucleotide is released in the presence of an enzymatic activity capable of cleaving said region of ribonucleotides.

74. The kit of claim 73, wherein said promoter oligonucleotide comprises said oligodeoxynucleotide.

75. The kit of claim 65, wherein an oligonucleotide which hybridizes to a region of said first DNA primer extension product and primes the synthesis of a second DNA primer extension product complementary to said first DNA primer extension product is not included in said kit.

76. The kit of claim 65 further comprising a reverse transcriptase.

77. The kit of claim 76, wherein said reverse transcriptase has an RNase H activity.

78. The kit of claim 76 further comprising an enzyme other than said reverse transcriptase which has an RNase H activity.

79. The kit of claim 65 further comprising a detection probe which hybridizes to said RNA target sequence or its complement to give a detectable signal during synthesis.

80. A composition comprising:

a promoter oligonucleotide having first and second regions, said first region comprising a 3′

-portion containing a contiguous arrangement of ribonucleotides and a portion 5′

thereto that is capable of hybridizing to a DNA template, and said second region being a promoter for an RNA polymerase, wherein said first region is situated 3′

to said second region, and wherein said promoter oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom; and

an oligodeoxynucleotide hybridized to said 3′

-portion of said first region, thereby forming an RNA;

DNA duplex.

81. A method for making available a promoter oligonucleotide having a 3′

-end capable of being extended in the presence of a DNA polymerase, said method comprising;

(a) providing said composition of claim 80 to a reaction mixture;

(b) exposing said composition to an enzyme capable of cleaving the ribonucleotides of said RNA;

DNA duplex, thereby releasing a blocking moiety situated at the 3′

-end of said promoter oligonucleotide and making available the remaining portion of said first region for hybridization to said DNA template and extension of a 3′

-end thereof in the presence of a DNA polymerase.

82. A method for making available a priming oligonucleotide for extension in the presence of a target nucleic acid, said method comprising the steps of:

(a) providing said priming oligonucleotide to a reaction mixture containing said target nucleic acid, said priming oligonucleotide having a cap hybridized to a region at the 3′

-end thereof, wherein the 5′

-terminal base of said cap is complementary to the 3′

-terminal base of said priming oligonucleotide, and wherein said cap is modified to prevent the initiation of DNA synthesis therefrom;

(b) hybridizing said priming oligonucleotide to a target sequence contained within said target nucleic acid, thereby displacing said cap from said priming oligonucleotide; and

(c) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to said target sequence.

83. A method of synthesizing multiple copies of a target sequence, said method comprising the steps of:

(a) treating a target nucleic acid comprising an RNA target sequence with;

(i) a priming oligonucleotide which hybridizes to the 3′

-end of said target sequence such that a primer extension reaction can be initiated therefrom; and

(ii) a binding molecule which binds to said target nucleic acid adjacent to or near the 5′

-end of said target sequence;

(b) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to said target sequence, said DNA primer extension product having a 3′

-end which is determined by said binding molecule and which is complementary to the 5′

-end of said target sequence;

(c) separating said DNA primer extension product from said target sequence using an enzyme which selectively degrades said target sequence;

(d) treating said DNA primer extension product with a promoter oligonucleotide comprising first and second regions, said first region hybridizing to a 3′

-region of said DNA primer extension product to form a promoter oligonucleotide;

DNA primer extension product hybrid, and said second region being a promoter for an RNA polymerase and situated 5′

to said first region, wherein any oligonucleotide provided in said method which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom;

(e) treating said DNA primer extension product with an extender oligonucleotide, said extender oligonucleotide hybridizing to a region of said DNA primer extension product 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

DNA primer extension product hybrid, such that an extender oligonucleotide;

DNA primer extension product hybrid is formed;

(f) extending the 3′

-end of said DNA primer extension product in said promoter oligonucleotide;

DNA primer extension product hybrid to add a sequence complementary to said second region of said promoter oligonucleotide; and

(g) transcribing from said promoter oligonucleotide;

DNA primer extension product hybrid multiple RNA products complementary to said DNA primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said RNA products are substantially identical to the base sequence of said target sequence.

84. A method of synthesizing multiple copies of a target sequence, said method comprising the steps of:

(a) treating a target nucleic acid comprising an RNA target sequence with a priming oligonucleotide which hybridizes to the 3′

-end of said target sequence, such that a primer extension reaction can be initiated therefrom;

(b) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a first DNA primer extension product having an undefined 3′

-end and comprising a base region complementary to said target sequence;

(c) separating said first DNA primer extension product from said target nucleic acid using an enzyme which selectively degrades that portion of said target nucleic acid which is complementary to said first DNA primer extension product;

(d) treating said first DNA primer extension product with a promoter oligonucleotide comprising first and second regions, said first region hybridizing to a 3′

-region of said first DNA primer extension product to form a promoter oligonucleotide;

first DNA primer extension product hybrid, and said second region being a promoter for an RNA polymerase and situated 5′

to said first region, wherein any oligonucleotide provided in said method which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom

(e) treating said first DNA primer extension product with an extender oligonucleotide, said extender oligonucleotide hybridizing to a region of said first DNA primer extension product 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

first DNA primer extension product hybrid, such that an extender oligonucleotide;

first DNA primer extension product hybrid is formed; and

(f) transcribing from said promoter oligonucleotide;

first DNA primer extension product hybrid multiple first RNA products complementary to at least a portion of said first DNA primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said first RNA products are substantially identical to the base sequence of said target sequence.

85. A method of synthesizing multiple copies of a target sequence, said method comprising the steps of:

(a) treating a target nucleic acid comprising a DNA target sequence with a promoter oligonucleotide comprising first and second regions, said first region hybridizing to the 3′

-end of said target sequence to form a promoter oligonucleotide;

target nucleic acid hybrid, and said second region being a promoter for an RNA polymerase and situated 5′

to said first region, wherein any oligonucleotide provided in said method which comprises a promoter for an RNA polymerase is modified to prevent the initiation of DNA synthesis therefrom, and wherein said target nucleic acid is not treated with a restriction endonuclease prior to step (a);

(b) treating said target nucleic acid with an extender oligonucleotide, said extender oligonucleotide hybridizing to a region of said target nucleic acid 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

target nucleic acid hybrid such that an extender oligonucleotide;

target nucleic acid hybrid is formed;

(c) transcribing from said promoter oligonucleotide;

target nucleic acid hybrid multiple first RNA products comprising a base region complementary to said target sequence using an RNA polymerase which recognizes said promoter and initiates transcription therefrom;

(d) treating one of said first RNA products with a priming oligonucleotide which hybridizes to a 3′

-region of said first RNA product such that a primer extension reaction can be initiated therefrom;

(e) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to at least a portion of said first RNA product, said DNA primer extension product having a 3′

-end which is complementary to the 5′

-end of said first RNA product;

(f) separating said DNA primer extension product from said first RNA product using an enzyme which selectively degrades said first RNA product;

(g) treating said DNA primer extension product with said promoter oligonucleotide to form a promoter oligonucleotide;

DNA primer extension product hybrid;

(h) treating said DNA primer extension product with an extender oligonucleotide, said extender oligonucleotide hybridizing to a region of said DNA primer extension product 3′

to said promoter oligonucleotide of said promoter oligonucleotide;

DNA primer extension product hybrid such that an extender oligonucleotide;

DNA primer extension product hybrid is formed; and

(i) transcribing from said promoter oligonucleotide;

DNA primer extension product hybrid multiple second RNA products complementary to said DNA primer extension product using said RNA polymerase, wherein the base sequences of said second RNA products are substantially complementary to the base sequence of said target sequence.

86. An oligonucleotide up to 50 bases in length and comprising at least 10 contiguous bases of a base sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

6, SEQ ID NO;

7, the complement of SEQ ID NO;

7, SEQ ID NO;

8, the complement of SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

11, SEQ ID NO;

12, the complement of SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

15, SEQ ID NO;

16, the complement of SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

19, SEQ ID NO;

20, the complement of SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

23, SEQ ID NO;

24, the complement of SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

27, SEQ ID NO;

29, SEQ ID NO;

30, the complement of SEQ ID NO;

30, SEQ ID NO;

31, the complement of SEQ ID NO;

31, SEQ ID NO;

32, the complement of SEQ ID NO;

32, and equivalent sequences thereof containing thymine bases substituted for uracil bases.