Capture moieties for nucleic acids and uses thereof

• US 20030113781A1
• Filed: 12/06/2002
• Published: 06/19/2003
• Est. Priority Date: 06/06/2000
• Status: Active Grant

• Current Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Original Assignee: Luminex Molecular Diagnostics Incorporated (DiaSorin S.p.A.)
• Inventors: Bortolin, Susan, Zastawny, Roman L.
• Granted Patent: US 7,230,092 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer

Abstract

A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe includes: a first nucleic acid sequence complementary to the target sequence (target-binding sequence); and a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex. In use, the target and target-bind sequence hybridize to form a duplex. A probe can be used to detect a molecule containing the target sequence, act as a primer for synthesis or amplification, etc.

Claims

1. A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe comprising:

(i) a first nucleic acid sequence complementary to the target sequence; and

(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and

wherein;

(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions.

2. A probe of claim 1 wherein the first nucleic acid sequence is at least six nucleic acid bases in length.

3. A probe of claim 2, wherein the first nucleic acid sequence is up to forty, or is up to thirty, or is up to twenty-six nucleic acid bases in length and/or wherein:

the first nucleic acid sequence is between six and forty, or between eight and thirty-five, or between ten and thirty, or between fifteen and thirty or between eighteen and twenty-eight, or between twenty and twenty-six nucleic acid bases in length.

4. A probe of claim 3 wherein the first nucleic acid sequence is twenty-four nucleic acid bases in length.

5. A probe of claim 1 or claim 2 wherein the second nucleic acid sequence is at least four nucleic acid bases in length.

6. A probe of claim 5 wherein the second nucleic acid sequence is up to fourteen nucleic acid bases in length, and preferably, wherein the second nucleic acid sequence is between five and twelve, or between six and twelve, or between seven and twelve, or between eight and twelve, or between nine and twelve, or is ten nucleic acid bases in length.

7. A probe of any preceding claim wherein the first and second nucleic acid sequences are located immediately adjacent to each other.

8. A probe of any preceding claim, wherein the second nucleic acid sequence is complementary to a first said portion of the first sequence, the first said portion being located such that when the intramolecular duplex is formed, a first hairpin structure having a single-stranded end comprising a second portion of the first sequence is formed, so as to act as a nucleation site of the target and first sequences.

9. A probe of claim 8 wherein said single stranded end is at least two nucleic acid bases in length.

10. A probe of any of claims 1 to 3 wherein a portion of the first nucleic acid sequence which is not complementary to the second nucleic acid sequence is located distal to the second nucleic acid sequence and is of sufficient length to act as a nucleation site for hybridization of the target and first sequences.

11. A probe of any preceding claim further comprising third and fourth nucleic acid sequences located at an end of the probe distal to the second nucleic sequence, complementary to each other and linked to each other so as to hybridize to form a second hairpin structure comprising a second intramolecular duplex under said set of conditions.

12. A probe of claim 11, wherein a portion of the first nucleic sequence which is not complementary to the second nucleic acid sequence is located distal to the second nucleic acid sequence and is of sufficient length to act as a nucleation site for hybridization of the target and first sequences, and is located to be adjacent the second intramolecular duplex.

13. A probe of claim 11, in combination with a said target sequence, wherein the target nucleic acid sequence is a terminal sequence of a target molecule such that upon formation of the intermolecular duplex, the intermolecular duplex and the second intramolecular duplex form a nicked duplex.

14. A probe of claim 11 wherein the first intramolecular duplex has a lower T_m than the second intramolecular duplex under said set of conditions.

15. A probe of any of claims 11 to 14, wherein the second intramolecular duplex is between three and thirty base pairs, or between three and twenty-five, or between six and twenty, or between ten and eighteen, or between fourteen and seventeen base pairs in length.

16. A probe of claim 15, wherein the second intramolecular duplex is sixteen base pairs in length.

17. A probe of any of claims 11 to 16 wherein the third and fourth nucleic acid sequences are connected to each other by a single stranded nucleic acid sequence up to twenty bases, or up to eighteen, or up to sixteen bases, or up to fourteen, or up to twelve, or up to ten, or up to eight, or up to six bases in length, and at least two bases in length.

18. A probe of any of claims 11 to 17 wherein the first intramolecular duplex is no longer than the second intramolecular duplex and the second intramolecular duplex has a G:

C content greater than that of the first intramolecular duplex.

19. A probe of any preceding claim in combination with a nucleic acid molecule comprising the target sequence.

29. A probe of any of claims 1 to 28, further comprising means for attaching the probe to a solid support.

30. A probe of claim 29, wherein said means for attaching the probe to a solid support is a biotinylated nucleotide.

31. A probe of any of claims 1 to 28 wherein the probe is attached to a solid support.

32. A probe of claim 31 wherein the probe is attached at a point outside of the nucleic acid sequence of the probe complementary to the target sequence.

33. A probe of claim 32, wherein said point of attachment is a modified nucleotide located within:

region A, region B, region C, region D, region F, or region G of the probe;

or wherein said point of attachment is a modified nucleotide located within;

region B, region D, region F, or region G of the probe;

or wherein said point of attachment is a modified nucleotide located within;

region B or region G of the probe;

or wherein said point of attachment is a modified nucleotide located within;

region B of the probe.

34. A probe of any preceding claim, further comprising means for detecting the probe when a target sequence is hybridized thereto.

35. A probe of any preceding claim in combination with a said molecule comprising a target nucleic acid sequence complementary to the nucleic acid of the probe complementary thereto.

36. A combination of claim 35, wherein the probe and said molecule comprising a target nucleic acid sequence complementary to the nucleic acid of the probe complementary thereto are provided separate from each other.

37. A combination of claim 35 or 36, wherein the said molecule comprising a target nucleic acid further comprises means for detection thereof.

43. A repertoire of probes comprising a plurality of probes of any of claims 1 to 18.

44. A repertoire of probes of claim 43, comprising at least five, or at least ten or at least fifteen, or at least twenty, or at least twenty-five, or at least thirty, or at least forty, or at least fifty, or at least sixty, or at least seventy, or at least eighty, or at least ninety said probes.

45. A repertoire of probes according claim 43 or 44, wherein the first nucleic acid sequence of each probe is the same in length as that of every other probe.

46. A composition comprising a mixture of a plurality of microparticles, each microparticle having probes selected from a repertoire of probes according to any of claims 43 to 45 attached thereto such that substantially all different probes of the repertoire are attached to different microparticles.

47. A composition according to claim 46 wherein each probe comprises the same number of nucleic acid bases.

49. A kit of claim 48, wherein each said probe is a probe of any of claims 1 to 18.

50. A kit of claim 49, further comprising a plurality of first molecules each having a target nucleic acid sequence complementary to that of a said probe.

52. A composition of claim 51, wherein each said probe is a probe of any of claims 1 to 18.

53. A composition of claim 52, further comprising a molecule comprising a plurality of said molecules comprising a target sequences, there being a molecule for each probe.

62. A repertoire of claim 61, wherein each complement comprises a probe according to any of claims 1 to 18.

64. A kit of claim 63, wherein each probe is a probe according to any of claims 1 to 18.

68. A method of detecting a molecule comprising a target nucleic acid sequence, the method comprising:

providing a sample that may contain the molecule,

providing a probe of claim 1, wherein the first nucleic acid sequence of (i) is complementary to the target sequence of the molecule;

exposing the sample and probe to said conditions of (ii) to cause formation of the intermolecular duplex of (iii);

detecting the absence or presence of the intermolecular duplex.

69. A method according to claim 68, wherein the probe is a probe of any of claims 1 to 18.

70. A method according to claim 68, wherein the probe and target molecule are as defined in claim 13.

71. A method according to claim 70, comprising the further step of exposing the sample and probe to an agent which stabilizes the nicked duplex.

72. A method according to claim 71, wherein the agent is a duplex binding ligand.

73. A method according to claim 72 wherein adjacent portions of the intermolecular duplex and the second intramolecular duplex are included in a sequence-specific duplex binding site formed, or wherein the terminal base pair of the intermolecular duplex adjacent the second intramolecular duplex is included in a sequence-specific duplex binding site, and wherein said duplex-binding ligand is specific for a said duplex binding site.

91. A method according to claim 74, wherein the nucleic acid moiety is a probe as defined in any of claims 1 to 18.

93. A method according to claim 75, wherein the each probe is a probe as defined in any of claims 1 to 35.

94. A process according to claim 83, wherein the each primer is a probe as defined in any of claims 1 to 35.

95. A method according to claim 87, wherein the each primer is a probe as defined in any of claims 135.

20. A unimolecular probe for detecting the presence or absence in a sample of a target molecule comprising a target nucleic acid, the probe comprising a molecule selected from the group consisting of:

A-B-C-D-E-F-G, D-E-F-G, and E-F-G, wherein;

(i) each of A, C, D, E and G is a nucleic sequence;

(ii) E and G are complementary to each other and covalently linked to each other by F so as to form a first intramolecular duplex under a defined set of conditions;

(iii)

(a) for the molecule E-F-G, the entirety of sequence E and at least a portion of F together form a nucleic acid sequence substantially complementary to the target nucleic acid sequence of the target molecule, and the molecule E-F-G and the target molecule hybridize with each other such that the probe and target molecule form an intermolecular duplex under said conditions;

(b) for each of the molecules A-B-C-D-E-F-G and D-E-F-G, (1) the entirety of sequence E and at least a portion of D, or (2) the entirety of sequence E and at least a portion of D and at least a portion of F, together form a nucleic acid sequence substantially complementary to the target nucleic acid sequence of the target molecule, and each of the molecules A-B-C-D-E-F-G and D-E-F-G, and the target molecule, hybridize with each other such that the probe and target molecule form an intermolecular duplex under said conditions;

; and

(c) A and C of the molecule A-B-C-D-E-F-G are complementary to each other and covalently linked to each other by B so as to form a second intramolecular duplex under said set of conditions.

21. A probe of claim 20, wherein the sequences of A and C of A-B-C of the molecule A-B-C-D-E-F-G are selected such that A-B-C forms a hairpin more stable than a hairpin formed by E-F-G of the molecule A-B-C-D-E-F-G.

22. A probe of claim 21, wherein the T_m of the intramolecular duplex of the more stable hairpin has a higher calculated T_m than the intramolecular duplex of the less stable duplex according to the equation:

T_m=[(number of A+T)×

2°

C.+(number of G+C)×

4°

C.]]

23. A probe of claim 21 the length of the duplex of the more stable hairpin exceeds the length of the duplex of the less stable hairpin by at least sixteen, or at least fourteen, or at least twelve, or at least ten, or at least eight, or at least six nucleic acid base pairs.

24. A probe of any of any of claims 20 to 23, wherein E is at least four nucleic acid bases in length.

25. A probe of any of claims 20 to 24, wherein said nucleic acid sequence of the probe complementary to the target sequence is at least six, or is at least eight, or is at least ten and/or up to fifty, or forty, or thirty nucleic acid bases in length.

26. A probe of claim 25, wherein said nucleic acid sequence of the probe complementary to the target sequence is between six and thirty, or between eight and twenty-eight, or between ten and twenty-eight, or between twelve and twenty-six, or between fourteen and twenty-six, or between sixteen and twenty-six, or between eighteen and twenty-six, or between twenty and twenty-six, or between twenty-two and twenty-six nucleic acid bases in length.

27. A probe of any of claims 20 to 26, wherein the at least a portion of F of (iii)(a) or of (iii)(b)(2) is a nucleation region at least three, or at least four, or at least five, or at least six and/or:

no more than nine, no more than eight, or no more than six, nucleic acid bases in length, and preferably wherein the region is five bases in length.

28. A probe of any of claims 20 to 26, wherein the at least a portion of D of (iii)(b)(1) or of (iii)(b)(2) is a nucleation region at least three, or at least four, or at least five, or at least six and/or:

no more than nine, no more than eight, or no more than six, nucleic acid bases in length, and preferably wherein the region is five bases in length.

38. A repertoire of populations of oligonucleotide tags, each tag of a population comprising a probe of claim 20, wherein for each tag in a population, the nucleic acid sequence substantially complementary to the target nucleic acid sequence of the target molecule for the tag is different from that of each tag in a different population.

39. A repertoire of probes comprising a plurality of populations of probes, each probe of each population being the same as each other probe of the population and being a probe of any of claims 20 to 28, wherein for each probe, said nucleic acid sequence of the probe complementary to a target sequence is the same length as that for every other probe of the repertoire.

40. A repertoire of probes according to claim 39, wherein for each probe of a said population, said nucleic acid sequence of the probe complementary to a target sequence varies from said sequence of every other probe of a different population, by at least two nucleic acids, or by at least three nucleic acids, or by at least four nucleic acids, or by at least five nucleic acids, or by at least six nucleic acids.

41. A repertoire of probes according to claim 39, wherein for each probe of a said population, said nucleic acid sequence of the probe complementary to a target sequence shares no more than 98 percent, or no more than 97 percent, or no more than 95 percent, or no more than 90 percent or no more than 85 percent, or no more than 80 percent or no more than 75 percent, or no more than 70 percent, or no more than 67 percent or no more than 65 percent or no more than 60 percent, or no more than 55 percent identity with every other said sequence of every other probe of a different population.

42. A repertoire of probes according to claim 40, wherein for each probe of a said population, said nucleic acid sequence of the probe complementary to a target sequence, the sequence is between 16 and 26 nucleotide bases in length and varies from said sequence of every other probe of a different population, by at least three nucleic acids.

92. A method according to claim 74, wherein the nucleic acid moiety is a probe as defined in any of claims 20 to 35.

48. A kit for determining the presence in a sample of a first molecule -comprising a target nucleic acid sequence, the kit comprising:

(1) a probe for determining the presence of the first molecule, the probe comprising;

(i) a first nucleic acid sequence complementary to the target sequence; and

(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and

wherein;

(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions;

(2) a second molecule comprising a control nucleic acid sequence, the control nucleic acid sequence being different from the target nucleic acid sequence; and

(3) a probe for determining the presence of the second molecule, the probe comprising;

(i) a third nucleic acid sequence complementary to the control sequence; and

(ii) a fourth nucleic acid sequence complementary to a portion of the third nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under said set of conditions; and

wherein;

(iii) hybridization of the control and third sequences to form an intermolecular duplex occurs under said set of conditions.

51. A composition comprising a mixture of a plurality of microparticles, each microparticle having a probe attached thereto for detection of a target nucleic acid sequence, each probe comprising a single molecule comprising:

(i) a first tag sequence complementary to the target sequence; and

(ii) a nucleic acid second sequence complementary to a portion of the first tag sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and

wherein;

(iii) hybridization of the target and tag sequences to form an intermolecular duplex occurs under said set of conditions.

54. The composition of claim 51 or claim 52 comprising at least ten different populations of microparticles, each microparticle of a population having a different probe attached thereto from that of a different population, each population of microparticles being for detection of a target nucleic acid sequence different from that of each other population.

55. The composition of claim 54 wherein the tag sequence of each said probe of a population is identical to the tag sequence of every other said probe attached to that microparticle.

56. The composition of claim 51 or claim 52, further comprising means for detecting the intermolecular duplex of (iii).

57. The composition of claim 51, wherein each said tag sequence is at least 10 nucleotides in length.

58. The composition of claim 57, wherein each said tag sequence is up to 60 nucleotides in length.

59. The composition of claim 58, wherein each said tag sequence is between 12 and 50 nucleotides in length., or is between 12 and 40, or between 12 and 30, or is between 16 and 30 or is between 20 and 30, or is between 23 and 28, or is 24 bases in length.

60. The composition of claim 51 wherein each tag sequence of each population is at least 10 nucleotides in length and the sequence of each tag sequence in a said population varies from the sequence of a said tag sequence in a different population by at least three bases.

61. A repertoire of oligonucleotide tag complements, each complement belonging to a population of like complements and being a unimolecular moiety comprising:

(i) a first complement sequence complementary to a target tag sequence; and

(ii) a nucleic acid second sequence complementary to a portion of the first complement sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and

wherein;

(iii) hybridization of the complement and tag sequences to form an intermolecular duplex occurs under said set of conditions;

wherein there are at least ten said populations of tag complements, each tag of a population having a said complement sequence different from the complement sequence of the tags in every other population in the repertoire.

63. A kit for sorting and identifying polynucleotides, the kit comprising:

a solid phase support having one or more spatially discrete regions, each such region having a uniform population of substantially identical probes covalently attached, wherein each probe is a unimolecular moiety comprising;

(i) a first tag sequence complementary to a target sequence of said polynucleotide to be identified; and

(ii) a nucleic acid second sequence complementary to a portion of the first tag sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and

wherein;

(iii) hybridization of the target and tag sequences to form an intermolecular duplex occurs under said set of conditions.

65. A kit of claim 63 or claim 64 wherein said one or more solid phase supports is a planar substrate and wherein said one or more spatially discrete regions is a plurality of spatially addressable regions.

66. A kit of claim 63 or claim 64 wherein each of said one or more solid phase supports is a microparticle.

67. A kit of any of claims 63 to 66 wherein said plurality is at least ten, or at least twenty, or at least thirty, or at least forty, or at least fifty, or at least sixty.

74. A method for detecting a single-stranded nucleic acid target sequence, comprising:

(a) providing a nucleic acid capture moiety comprising a unimolecular moiety comprising;

(i) a first nucleic acid sequence complementary to the target sequence; and

(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and

wherein;

(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions;

(b) forming a reaction mixture comprising the single-stranded target nucleic acid and nucleic acid capture moiety under conditions such that the duplex of (a)(iii) forms;

(c) detecting the presence or absence of the duplex of (b);

such that the target single-stranded nucleic acid is detected.

75. A method of amplifying a nucleic acid molecule, the method comprising:

(a) providing a sample containing the nucleic acid molecule to be amplified;

(b) providing first and second probes, wherein;

(i) the first probe comprises a unimolecular capture moiety comprising;

(1) a forward primer; and

(2) a nucleic acid sequence complementary to a portion of the forward primer and capable of hybridization therewith to form a first intramolecular duplex under a set of conditions suitable for amplifying the nucleic acid molecule;

(ii) the second probe comprises a unimolecular capture moiety comprising;

(3) a reverse primer, and

(4) a nucleic acid sequence complementary to a portion of the reverse primer and capable of hybridization therewith to form a first intramolecular duplex under said set of conditions;

wherein;

(c) exposing the sample and first and second probes to said conditions so as to amplify the nucleic acid molecule.

76. A method of synthesizing a nucleic acid molecule, the method comprising:

(a) providing a sample containing or suspected of containing the nucleic acid to be synthesized;

(b) providing a probe comprising a unimolecular capture moiety comprising;

(1) a first nucleic acid sequence complementary to a sequence of the nucleic acid molecule to be synthesized so as to act as primer for said synthesis; and

(2) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a set of conditions suitable for said synthesis; and

(c) exposing the sample and probe to said conditions so as to synthesize the nucleic acid molecule.

77. A method of detecting first and second target sequences comprising first and second target nucleic acid sequences, the method comprising:

(a) providing a sample that may contain the first and second sequences;

(b) providing first and second probes, wherein;

(i) the first probe comprises a unimolecular capture moiety comprising;

(1) a first nucleic acid sequence complementary to the first target sequence; and

(2) a nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form an intramolecular duplex therewith under a defined set of conditions;

(ii) the second probe comprises a unimolecular capture moiety comprising;

(3) a second nucleic acid sequence complementary to the second target sequence; and

(4) a nucleic acid sequence complementary to a portion of the second nucleic acid sequence and capable of hybridization therewith to form an intramolecular duplex therewith under said set of conditions;

wherein;

(iii) hybridization of the first target and first nucleic acid sequences to form a first intermolecular duplex occurs under said set of conditions, and hybridization of the second target and second nucleic acid sequences to form a second intermolecular duplex occurs under said set of conditions; and

(iv) the first and second target sequences differ from each other by at least one nucleotide base; and

(c) detecting the absence or presence of each of the first and second intermolecular duplexes.

78. A method according to claim 77 wherein the first and second target sequences are of equal nucleotide base length.

79. A method according to claim 77 or 78, wherein, for the first probe, a nucleotide base in a terminal position of the nucleic acid sequence complementary to a portion of the first nucleic acid sequence is complementary to the nucleotide base which hybridizes to the at least one nucleotide base of (b)(iv).

80. A method according to claim 79, wherein, for the second probe, a nucleotide base in a terminal position of the nucleic acid sequence complementary to a portion of the first nucleic acid sequence is complementary to the nucleotide base which hybridizes to the at least one nucleotide base of (b)(iv).

81. A method according to any of claims 77 to 80 wherein the first and second target sequences vary from each other by only the one nucleotide base of (b)(iv).

82. A method of any of claims 77 to 80, wherein the first and second target sequences vary from each other by at least two, or at least three nucleotide bases.

83. A process for amplifying nucleic acids comprising:

(1) providing a nucleic acid template;

(2) hybridizing at least one primer to the 3′

-end of the template, wherein the primer comprises;

(i) a nucleic acid sequence complementary to said 3′

-end of the template and having a free 3′

-hydroxyl;

(ii) a nucleic acid sequence complementary to a portion of the nucleic acid sequence of (i) and capable of hybridization therewith to form a first intramolecular duplex under a set of conditions suitable for amplifying the nucleic acid molecule; and

(3) amplifying the primer-template hybrid using at least one temperature-stable polymerase enzyme to produce linear amplification products.

84. A process of claim 83, wherein the primer further comprises means located upstream of the nucleic acid sequence complementary to the 3′

-end of the template for precluding synthesis therepast of a nucleic acid molecule complementary to the primer upstream of said means.

85. A process of claim 84, wherein said means is a modified nucleotide.

86. A process of claim 970, wherein the primer further comprises a modified nucleotide located immediately upstream of the nucleic acid sequence complementary to the 3′

-end of the template so as to preclude extension of the 3′

-end of the template.

87. A method of diagnosing a disease comprising:

(1) obtaining a nucleic acid template from a biological sample;

(2) mixing, under a defined set of condition, at least one primer, specific for the disease associated with the expression of one or more genes, with the sample, the primer comprising;

(i) a first nucleic acid sequence complementary to the template; and

(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under the set of conditions; and

wherein;

(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions; and

(3) detecting the presence or absence of the intermolecular duplex of (2)(iii) in order to determine the presence or absence of the template in the biological sample of (1).

88. A method of diagnosing a disease comprising:

(1) obtaining a nucleic acid template from a biological sample;

(2) mixing at least one primer, specific for the disease associated with the expression of one or more genes, with the sample to form a primer-template hybrid, the primer comprising;

(i) a nucleic acid sequence complementary to a 3′

-end of the template and having a free 3′

-hydroxyl;

(ii) a nucleic acid sequence complementary to a portion of the nucleic acid sequence of (i) and capable of hybridization therewith to form an intramolecular duplex under conditions in which the primer-template hybrid forms; and

(3) amplifying the primer-template hybrid using at least one temperature-stable polymerase enzyme to produce amplification products.

89. A method of claim 88, comprising the further step of diagnosing said diseases by detecting the presence, absence or the amount of amplified nucleic acid sequences present in said biological sample.

90. A method of claim 89 wherein step (3) is a linear amplification.