Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases

Claims

We claim:

1. A method of amplifying a nucleic acid segment, comprising the steps of:

(a) providing a nucleic acid segment which comprises at least one 2'-deoxyribonucleotide or 2'-deoxyribonucleotide analog, said segment having the sequence of an RNA which can be autocatalytically replicated by the DNA-Dependent RNA polymerase activity of an RNA replicase; and

(b) subjecting said segment to conditions effective for autocatalytic replication by said replicase.

2. The method of claim 1, wherein fewer than 10% of the nucleotides of said segment are 2'-deoxyribonucleotide analogs or ribonucleotide analogs.

3. The method of claim 2 wherein said segment consists of 2'-deoxyribonucleotides, 2'-deoxyribonucleotide phosphate analogs or ribonucleotides.

4. The method of claim 3 wherein said segment consists of 2'-deoxyribonucleotides or ribonucleotides.

5. The method of claim 4 wherein said segment is a DNA segment.

6. The method of claim 5 wherein said segment is a segment of a DNA.

7. The method of claim 1 wherein said segment is a segment of a single-stranded nucleic acid.

8. The method of claim 4 wherein said segment is a segment of a single-stranded nucleic acid.

9. The method of claim 5 wherein said segment is a segment of a single-stranded nucleic acid.

10. The method of claim 6 wherein said segment is a segment of a single-stranded DNA.

11. The method of claim 1 wherein the nucleic acid segment is a segment of a strand of a double-stranded or partially double-stranded nucleic acid.

12. The method of claim 4 wherein the nucleic acid segment is a segment of a strand of a double-stranded or partially double-stranded nucleic acid.

13. The method of claim 5 wherein the nucleic acid segment is a segment of a strand of a double-stranded or partially double-stranded nucleic acid.

14. The method of claim 6 wherein the nucleic acid segment is a segment of a strand of a double-stranded or partially double-stranded nucleic acid.

15. The method of claim 14 wherein both strands of the double-stranded or partially double-stranded nucleic acid are DNAs.

16. The method according to any one of claims 1-15 wherein the replicase is QB replicase.

17. The method according to claim 16 wherein the conditions for effective for autocatalytic replication further comprise having, in a solution in which the replication occurs, an ion selected from the group consisting of Mn.sup.+2, Co.sup.+2, and Zn.sup.+2 at a concentration greater than 0.5 mM.

18. The method according to claim 17 wherein the ion is selected from the group consisting of Mn.sup.+2 and Co.sup.+2 and is present at a concentration between about 0.5 mM and 5 mM.

19. A method of treating a nucleic acid sample to make reporter RNA, comprising the steps of:

(a) providing a nucleic acid sample comprising a target segment;

(b) providing a probe consisting of probe molecules, each probe molecule comprising:

(i) an anti-target segment capable of hybridizing to the target segment; and

(ii) an amplifiable complex nucleic acid segment, having a sequence, or a sequence which is fully complementary and of the same length, of a reporter RNA sequence which is autocatalytically replicatable by an RNA replicase;

(c) combining the nucleic acid sample with the probe molecules under conditions suitable for hybridization, such that at least one probe molecule anti-target segment hybridizes to the nucleic acid sample target segment with sufficient stringency such that the hybridized anti-target segment will not be removed in step (d);

(d) removing any unhybridized probe molecules from all hybridized probe molecules;

(e) subjecting the complex nucleic acid segment of each hybridized probe molecule to conditions effective for catalysis with the DNA-dependent RNA polymerase activity of the replicase resulting in autocatalytic replication of the complex nucleic acid segment to make reporter RNA.

20. The method of claim 19 wherein the probe is DNA and the amplifiable complex nucleic acid segment is attached to the 5'-terminus of the anti-target segment and step (d) further comprises exonuclease digestion of unhybridized probes by an exonuclease having 3'- to 5'-single-stranded nuclease activity.

21. The method according to claim 19, wherein the complex nucleic acid segment has a sequence of a nanovariant DNA or a midivariant DNA.

22. The method according to claim 19, further comprising the step of assaying for the reporter RNA.

23. The method according to claim 22, wherein the assay for reporter RNA comprises staining all RNA obtained in step (e) to identify reporter RNA.

24. The method according to claim 23, wherein the assay for reporter RNA further comprises separating by size all resulting RNA in step (e) and determining whether stained RNA has the size of reporter RNA.

25. The method according to claim 22, wherein the assay further comprises:

(1) adding a radiolabelled ribonucleoside triphosphate, or ribonucleoside triphosphate analog having a derivatized base, in step (e) during autocatalytic replication for incorporation into the reporter RNA to produce labelled reporter RNA;

(2) separating the labelled reporter RNA from unincorporated labelled ribonucleoside triphosphate or unincorporated labelled ribonucleoside triphosphate analog; and

(3) detecting the labelled reporter RNA.

26. The method according to claim 25, wherein the label is a radiolabel selected from the group consisting of .sup.32 P and .sup.35 S.

27. The method according to claim 25, wherein the ribonucleoside triphosphate analog is uracil derivatized at carbon-5 with a label selected from the group consisting of biotin, iminobiotin and digoxigenin.

28. The method according to claim 22, wherein the assay comprises a nucleic acid probe hybridization assay for the presence of reporter RNA.

29. The method according to claim 22, wherein the assay comprises measuring by bioluminescence whether ATP is depleted by autocatalytic replication in step (e).

30. The method of claim 19 wherein the conditions for effective for autocatalytic replication further comprise having, in a solution in which the replication occurs, an ion selected from the group consisting of Mn.sup.+2, co.sup.+2, and Zn.sup.+2 at a concentration greater than 0.5 Mm.

31. The method according to claim 19 wherein the ion is selected from the group consisting of Mn.sup.+2 and Co.sup.+2 and is present at a concentration between about 0.5 Mm and 5 Mm.

32. The method according to claim 19, wherein the replicase is QB replicase.

33. A method of treating a nucleic acid sample to make reporter RNA, comprising the steps of:

(a) providing a nucleic acid sample comprising a target segment;

(b) providing a first probe comprising:

(i) a first anti-target subsegment capable of hybridizing to a portion of the target segment; and

(ii) a first portion of an amplifiable segment;

(c) providing a second probe comprising:

(i) a second anti-target subsegment capable of hybridizing to a portion of the target segment; and

(ii) a second portion of the amplifiable segment, the first and second portions together having a sequence, or a sequence which is fully complementary and of the same length, of a reporter RNA sequence which is autocatalytically replicatable by an RNA replicase;

(d) combining the nucleic acid sample with the first and second probes under conditions suitable for hybridization, such that both the first anti-target subsegment and the second anti-target subsegment hybridize to the target segment with sufficient stringency to allow the first and second anti-target subsegments to be processed in step (e) to form a complex or broken complex;

(e) processing the hybridized first and second probes to create the broken complex nucleic acid segment or the complex nucleic acid segment, the complex nucleic acid segment comprising the amplifiable segment with one or more nucleotides inserted therein;

(f) subjecting the nucleic acid segment of step (e) to conditions effective for catalysis with the DNA-dependent RNA polymerase activity of the replicase resulting in autocatalytic replication of the nucleic acid segment to make reporter RNA.

34. The method according to claim 33, wherein step (e) further comprises forming a complex nucleic acid segment by ligating the broken complex nucleic acid segment.

35. The method according to claim 33, wherein the hybridized first and second anti-target subsegments are separated by a gap of at least one nucleotide, and step (e) further comprises extending one of the anti-target subsegments in a primer extending reaction to fill in the gap and create a broken complex nucleic acid segment.

36. The method according to claim 35, further comprising forming a complex nucleic acid segment by ligating the broken complex nucleic acid segment.

37. The method according to claim 33, wherein the complex nucleic acid segment or broken complex nucleic acid segment has a sequence of a nanovariant DNA or a midivariant DNA into which a sequence consisting of the sequence of the anti-target subsegments is inserted.

38. The method according to claim 35, wherein the complex nucleic acid segment or broken complex nucleic acid segment has a sequence of a nanovariant DNA or a midivariant DNA into which a sequence consisting of the sequence of the anti-target subsegments and the sequence of the anti-target subsegment extension is inserted.

39. The method according to claim 33, further comprising the step of assaying for the reporter RNA.

40. The method according to claim 39, wherein the assay for reporter RNA comprises staining all RNA obtained in step (f) to identify reporter RNA.

41. The method according to claim 40, wherein the assay for reporter RNA further comprises separating by size all resulting RNA in step (f) and determining whether stained RNA has the size of reporter RNA.

42. The method according to claim 39, wherein the assay further comprises:

(1) adding a radiolabelled ribonucleoside triphosphate, or ribonucleoside triphosphate analog having a derivatized base, in step (f) during autocatalytic replication for incorporation into the reporter RNA to produce labelled reporter RNA;

(2) separating the labelled reporter RNA from unincorporated labelled ribonucleoside triphosphate or unincorporated labelled ribonucleoside triphosphate analog; and

(3) detecting the labelled reporter RNA.

43. The method according to claim 42, wherein the label is a radiolabel selected from the group consisting of .sup.32 P and .sup.35 S.

44. The method according to claim 42, wherein the ribonucleoside triphosphate analog is uracil derivatized at carbon-5 with a label selected from the group consisting of biotin, iminobiotin and digoxigenin.

45. The method according to claim 42, wherein the assay comprises a nucleic acid probe hybridization assay for the presence of reporter RNA.

46. The method according to claim 42, wherein the assay comprises measuring by bioluminescence whether ATP is depleted by autocatalytic replication in step (f).

47. The method of claim 33 wherein each probe is DNA.

48. The method of claim 33 wherein the conditions for effective for autocatalytic replication further comprise having, in a solution in which the replication occurs, an ion selected from the group consisting of Mn.sup.+2, Co.sup.+2, and Zn.sup.+2 at a concentration greater than 0.5 Mm.

49. The method according to claim 48 wherein the ion is selected from the group consisting of Mn.sup.+2 and Co.sup.+2 and is present at a concentration between about 0.5 Mm and 5 Mm.

50. The method according to claim 33, wherein the replicase is QB replicase.

51. A method of treating a nucleic acid sample to make reporter RNA, comprising the steps of:

(a) providing a nucleic acid sample comprising a first target segment;

(b) providing a first probe comprising:

(i) a first anti-target segment capable of hybridizing to the first target segment; and

(ii) a first portion of an amplifiable segment;

(c) providing a second probe comprising:

(i) a second anti-target segment capable of hybridizing to a second target segment; and

(ii) a second portion of the amplifiable segment, the first portion and the complement of the second portion together having a sequence, or a sequence which is fully complementary and of the same length, of a reporter RNA sequence which is autocatalytically replicatable by an RNA replicase;

(d) combining the nucleic acid sample with the first and second probes under conditions suitable for hybridization, such that the first anti-target segment of the first probe hybridizes to the first target segment with sufficient stringency so as to allow the first probe to act as a primer in step (e);

(e) extending the hybridized first probe in a first primer extending reaction using the hybridized nucleic acid sample as a template to produce the second target segment adjacent to the 3' end of the first probe;

(f) separating the hybridized strands of the extended first probe and the nucleic acid sample;

(g) hybridizing the second anti-target segment of the second probe to the second target segment of the first probe with sufficient stringency so as to allow the second probe to act as a primer in step (h);

(h) extending the hybridized second probe in a second primer extending reaction using the hybridized first probe as a template to produce a complex nucleic acid segment, the complex nucleic acid comprising the amplifiable segment with one or more nucleotides inserted therein; and

(i) subjecting the complex nucleic acid segment to conditions effective for catalysis with the DNA-dependent RNA polymerase activity of the replicase resulting in autocatalytic replication of the complex nucleic acid segment to make reporter RNA.

52. The method according to claim 51, wherein the extension of the probes is catalyzed by a reverse transcriptase.

53. The method according to claim 52, wherein the reverse transcriptase is selected from the group consisting of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase.

54. The method according to claim 51, wherein both probes are DNA.

55. The method according to claim 51, further comprising the step of assaying for the reporter RNA.

56. The method according to claim 55, wherein the assay for reporter RNA comprises staining all RNA obtained in step (i) to identify reporter RNA.

57. The method according to claim 56, wherein the assay for reporter RNA further comprises separating by size all resulting RNA in step (i) and determining whether stained RNA has the size of reporter RNA.

58. The method according to claim 55, wherein the assay further comprises:

(1) adding a radiolabelled ribonucleoside triphosphate, or ribonucleoside triphosphate analog having a derivatized base, in step (i) during autocatalytic replication for incorporation into the reporter RNA to produce labelled reporter RNA;

(2) separating the labelled reporter RNA from unincorporated labelled ribonucleoside triphosphate or unincorporated labelled ribonucleoside triphosphate analog; and

(3) detecting the labelled reporter RNA.

59. The method according to claim 58, wherein the label is a radiolabel selected from the group consisting of .sup.32 P and 35S.

60. The method according to claim 58, wherein the ribonucleoside triphosphate analog is uracil derivatized at carbon-5 with a label selected from the group consisting of biotin, iminobiotin and digoxigenin.

61. The method according to claim 55, wherein the assay comprises a nucleic acid probe hybridization assay for the presence of reporter RNA.

62. The method according to claim 55, wherein the assay comprises measuring by bioluminescence whether ATP is depleted by autocatalytic replication in step (i).

63. The method of claim 51 wherein the conditions for effective for autocatalytic replication further comprise having, in a solution in which the replication occurs, an ion selected from the group consisting of Mn.sup.+2 Co.sup.+2, and Zn.sup.+2 at a concentration greater than 0.5 Mm.

64. The method according to claim 63 wherein the ion is selected from the group consisting of Mn.sup.+2 and Co.sup.+2 and is present at a concentration between about 0.5 Mm and 5 Mm.

65. The method according to claim 51, wherein the replicase is QB replicase.

66. A method of treating a nucleic acid sample to make reporter RNA, comprising the steps of:

(a) providing a nucleic acid sample comprising a target segment;

(b) providing an RNA probe comprising:

(i) an anti-target segment which is capable of hybridizing to the target segment; and

(ii) an amplifiable segment having a sequence, or sequence which is fully complementary and of the same length, of a reporter RNA sequence which is autocatalytically replicatable by an RNA replicase, the amplifiable segment adjacent to the 5' end of the anti-target segment;

(c) combining the nucleic acid sample with the probe under conditions suitable for hybridization, such that the anti-target segment hybridizes to the target segment with sufficient stringency so as to allow the probe to act as a template in step (e);

(d) treating the nucleic acid sample, prior to or following step (c), so that at least the target segment hybridized to the probe is the 3'-terminal segment of the nucleic acid sample and has a hydroxyl group at its 3'-terminus;

(e) extending the hybridized nucleic acid sample in an primer extending reaction using the probe as a template to produce a complex nucleic acid adjacent to the 3' end of the target segment, the complex nucleic acid having a sequence complementary to the amplifiable segment;

(f) digesting the RNA probe either chemically or enzymatically;

(g) inactivating the chemical or enzyme used for digestion; and

(h) subjecting the complex nucleic acid segment to conditions effective for catalysis with the DNA-dependent RNA polymerase activity of the replicase resulting in autocatalytic replication of the complex nucleic acid to make reporter RNA.

67. The method according to claim 66, wherein the RNA probes are digested by treatment with a base.

68. The method according to claim 66, wherein the RNA probes are digested by treatment with a ribonuclease.

69. The method according to claim 66, wherein the extension of the nucleic acid sample is catalyzed by a reverse transcriptase.

70. The method according to claim 69, wherein the reverse transcriptase is selected from the group consisting of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase.

71. The method according to claim 66, further comprising the step of assaying for the reporter RNA.

72. The method according to claim 71, wherein the assay for reporter RNA comprises staining all RNA obtained in step (h) to identify reporter RNA.

73. The method according to claim 72, wherein the assay for reporter RNA further comprises separating by size all resulting RNA in step (h) and determining whether stained RNA has the size of reporter RNA.

74. The method according to claim 71, wherein the assay further comprises:

(1) adding a radiolabelled ribonucleoside triphosphate or ribonucleoside triphosphate analog having a derivatized base, in step (h) during autocatalytic replication for incorporation into the reporter RNA to produce labelled reporter RNA;

(2) separating the labelled reporter RNA from unincorporated labelled

ribonucleoside triphosphate or unincorporated labelled ribonucleoside triphosphate analog; and

(3) detecting the labelled reporter RNA.

75. The method according to claim 74, wherein the label is a radiolabel selected from the group consisting of .sup.32 P and 35S.

76. The method according to claim 74, wherein the ribonucleoside triphosphate analog is uracil derivatized at carbon-5 with a label selected from the group consisting of biotin, iminobiotin and digoxigenin.

77. The method according to claim 71, wherein the assay comprises a nucleic acid probe hybridization assay for the presence of reporter RNA.

78. The method according to claim 71, wherein the assay comprises measuring by bioluminescence whether ATP is depleted by autocatalytic replication in step (h).

79. The method of claim 66 wherein the conditions for effective for autocatalytic replication further comprise having, in a solution in which the replication occurs, an ion selected from the group consisting of Mn.sup.+2, Co.sup.+2, and Zn.sup.+2 at a concentration greater than 0.5 Mm.

80. The method according to claim 79 wherein the ion is selected from the group consisting of Mn.sup.+2 and Co.sup.+2 and is present at a concentration between about 0.5 Mm and 5 Mm.

81. The method according to claim 66, wherein the replicase is QB replicase.

82. A method of treating a nucleic acid sample to make reporter RNA, comprising the steps of:

(a) providing a nucleic acid sample comprising first and second target segments, the first target segment located at a position 5' to the second target segment, the target segments separated by at least one nucleotide;

(b) providing a first probe comprising a first anti-target segment capable of hybridizing to the first target segment;

(c) providing a second probe comprising a second anti-target segment capable of hybridizing to the second target segment;

(d) combining the nucleic acid sample with the first and second probes under conditions suitable for hybridization, such that the first probe hybridizes to first target segment with sufficient stringency so as to act as a physical barrier to extension of the second probe, and the second probe hybridizes to the second target segment with sufficient stringency so as to allow the second probe to act as a primer in step (e);

(e) extending the hybridized second probe in a first primer extending reaction using the hybridized nucleic acid sample as a template to produce a third target segment adjacent to the 3' end of the second probe, such that the 3' end of the extended second probe adjoins the 5' end of the first hybridized probe;

(f) separating the hybridized strands of the extended second probe and the nucleic acid sample;

(g) providing a third RNA probe comprising:

(i) a third anti-target segment; and

(ii) an amplifiable segment having a sequence, or sequence which is fully complementary and of the same length, of a reporter RNA sequence which is autocatalytically replicatable by an RNA replicase, the amplifiable segment adjacent to the 5' end of the anti-target segment;

(h) combining the extended second probe with the third RNA probe under conditions suitable for hybridization, such that the third probe hybridizes to the third target segment of the extended second probe with sufficient stringency so as to allow the third probe to act as a template in step (i);

(i) extending further the hybridized extended second probe in a second primer extending reaction using the amplifiable segment of the third probe for a template to produce a complex nucleic acid segment adjacent to the 3' end of the third target segment, the complex nucleic acid segment having a sequence complementary to the amplifiable segment;

(j) digesting both hybridized and unhybridized RNA probes either chemically or enzymatically;

(k) inactivating the chemical or enzyme used for digesting; and

(l) subjecting the complex nucleic acid segment to conditions effective for catalysis with the DNA-dependent RNA polymerase activity of the replicase resulting in autocatalytic replication of the complex nucleic acid segment to make reporter RNA.

83. The method according to claim 82, wherein the third anti-target segment of the third probe is at least 12 nucleotides in length and has the sequence fully complementary and of the same length to that of the third target segment of the extended second probe and wherein there are at least 12 nucleotides between the target segments in the nucleic acid sample.

84. The method according to claim 82, wherein the third RNA probe is digested by treatment with a base.

85. The method according to claim 82, wherein the third RNA probe is digested by treatment with a ribonuclease.

86. The method according to claim 82, wherein the extension of the nucleic acid sample is catalyzed by a reverse transcriptase.

87. The method according to claim 86, wherein the reverse transcriptase is selected from the group consisting of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase.

88. The method according to claim 82, further comprising the step of assaying for the reporter RNA.

89. The method according to claim 88, wherein the assay for reporter RNA comprises staining all RNA obtained in step (l) to identify reporter RNA.

90. The method according to claim 89, wherein the assay for reporter RNA further comprises separating by size all resulting RNA in step (l) and determining whether stained RNA has the size of reporter RNA.

91. The method according to claim 88, wherein the assay further comprises:

(1) adding a radiolabelled ribonucleoside triphosphate, or ribonucleoside triphosphate analog having a derivatized base, in step (l) during autocatalytic replication for incorporation into the reporter RNA to produce labelled reporter RNA;

(2) separating the labelled reporter RNA from unincorporated labelled ribonucleoside triphosphate or labelled ribonucleoside triphosphate analog; and

(3) detecting the labelled reporter RNA.

92. The method according to claim 91, wherein the label is a radiolabel selected from the group consisting of .sup.32 P and .sup.35 S.

93. The method according to claim 91, wherein the ribonucleoside triphosphate analog is uracil derivatized at carbon-5 with a label selected from the group consisting of biotin, iminobiotin and digoxigenin.

94. The method according to claim 88, wherein the assay comprises a nucleic acid probe hybridization assay for the presence of reporter RNA.

95. The method according to claim 88, wherein the assay comprises measuring by bioluminescence whether ATP is depleted by autocatalytic replication in step (l).

96. The method of claim 82 wherein the conditions for effective for autocatalytic replication further comprise having, in a solution in which the replication occurs, an ion selected from the group consisting of Mn.sup.+2, Co.sup.+2, and Zn.sup.+2 at a concentration greater than 0.5 Mm.

97. The method according to claim 96 wherein the ion is selected from the group consisting of Mn.sup.+2 and Co.sup.+2 and is present at a concentration between about 0.5 Mm and 5 Mm.

98. The method according to claim 82, wherein the replicase is QB replicase.

99. A test kit for amplifying reporter RNA, the kit comprising:

(a) an RNA replicase having DNA-dependent RNA polymerase activity;

(b) a probe comprising:

(i) an anti-target segment, the anti-target segment capable of hybridizing to a target segment in a nucleic acid sample with sufficient stringency to allow the probe to act as a template for autocatalytic replication; and

(ii) an amplifiable segment having a sequence, or sequence which is fully complementary and of the same length, of a reporter RNA sequence which is autoca talytically replicatable by the RNA replicase;

wherein the probe is a nucleic acid which comprises at least one 2'-deoxyribonucleotide or one 2'-deoxyribonucleotide analog residue.

100. The test kit according to claim 99 further comprising an exonuclease having 3'- to 5'-single-stranded nuclease activity wherein the amplifiable complex segment is attached to the 5'-terminus of t