Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon

• US 6,090,552 A
• Filed: 07/11/1997
• Issued: 07/18/2000
• Est. Priority Date: 07/16/1996
• Status: Expired due to Term

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: Intergen Company
• Inventors: Bhatnagar, Satish K., Nazarenko, Irina A., Hohman, Robert J., Winn-Deen, Emily S.
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/891,516
• Time in Patent Office: 1,103 Days
• Field of Search: 435/6, 435/91.2, 536/24.33, 536/24.3, 536/25.32, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/113 Telomerase
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

The present invention provides labeled nucleic acid amplification oligonucleotides, which can be linear or hairpin primers or blocking oligonucleotides. The oligonucleotides of the invention are labeled with donor and/or acceptor moieties of molecular energy transfer pairs. The moieties can be fluorophores, such that fluorescent energy emitted by the donor is absorbed by the acceptor. The acceptor may be a fluorophore that fluoresces at a wavelength different from the donor moiety, or it may be a quencher. The oligonucleotides of the invention are configured so that a donor moiety and an acceptor moiety are incorporated into the amplification product. The invention also provides methods and kits for directly detecting amplification products employing the nucleic acid amplification primers. When labeled linear primers are used, treatment with exonuclease or by using specific temperature eliminates the need for separation of unincorporated primers. This "closed-tube" format greatly reduces the possibility of carryover contamination with amplification products, provides for high throughput of samples, and may be totally automated.

Claims

1. A method of detecting telomerase activity comprising:

(a) contacting a sample suspected of having telomerase activity with at least two oligonucleotide primers comprising a first primer and a second primer, wherein said first primer comprises the following continuous sequences in 5'"'"' to 3'"'"' order;

(i) a first nucleotide sequence of 6-30 nucleotides, wherein a nucleotide within said first nucleotide sequences is labeled with a first moiety selected from the group consisting of a donor moiety and an acceptor moiety of a molecular energy transfer pair, wherein the donor moiety emits energy of one or more particular wavelengths when excited, and the acceptormoiety absorbs energy at one or more particular wavelengths emitted by the donor moiety;

(ii) a second, single-stranded nucleotide sequence of 3-20 or nucleotides;

(iii) a third nucleotide sequence of 6-30 nucleotides, wherein a nucleotides within said third nucleotide sequence is labeled with a second moiety selected from the group consisting of said donor moiety and said acceptor moiety, and said second moiety is the member of said group not labeling said first nucleotide sequence, wherein said third nucleotide sequence is sufficiently complementary in reverse order to said first nucleotide sequence for a duplex to form between said first nucleotide sequence and said third nucleotide sequence such that said first moiety and second moiety are in sufficient proximity such that, when the donor moiety is excited and emits energy, the acceptor moiety absorbs energy emitted by the donor moiety; and

(iv) at the 3'"'"' end of said first primer, a fourth, single-stranded nucleotide sequence of 8-40 nucleotides that comprises at its 3'"'"' end a sequence that is a substrate for a telomerase;

wherein said second primer comprises at its 3'"'"' end a sequence sufficiently complementary so as to be able to hybridize to telomeric repeats that result from the activity of said telomerase;

(b) subjecting the sample to conditions suitable for telomerase activity;

(c) conducting a nucleic acid amplification reaction under conditions suitable for said first and second primers to prime DNA synthesis;

(d) stimulating energy emission from said donor moiety; and

(e) detecting or measuring energy emitted by said donor moiety or acceptor moiety, the presence or amount of said energy indicating the presence or amount of telomerase activity in the sample.

2. A method for determining if a target nucleotide sequence is present in a sample comprising:

(a) contacting the sample with an oligonucleotide containing;

(i) a first nucleotide sequence,

(ii) a second nucleotide sequence at the 5'"'"' end of the first nucleotide sequence,

(iii) a third nucleotide sequence at the 5'"'"' end of the second nucleotide sequence, and

(iv) a fourth nucleotide sequence at the 5'"'"' end of the third nucleotide sequence,

wherein the oligonucleotide is capable of forming a hairpin containing nucleotides of the second and fourth nucleotide sequences, and the oligonucleotide emits a detectable signal if the hairpin is not formed,

(b) incorporating the oligonucleotide into a double-stranded nucleic acid, and

(c) determining that the target nucleotide sequence is present in the sample if the signal is detected, or determining that the target nucleotide sequence is not present in the sample if the signal is not detected.

3. The method of claim 2, wherein the first nucleotide sequence contains the nucleotide at the 3'"'"' end of the oligonucleotide, the fourth nucleotide sequence contains the nucleotide at the 5'"'"' end of the oligonucleotide, and the first nucleotide sequence is not complementary to the fourth nucleotide sequence.

4. The method of claim 3, wherein the first nucleotide sequence is not complementary to the second nucleotide sequence.

5. The method of claim 3, wherein the first nucleotide sequence is not complementary to the third nucleotide sequence.

6. The method of claim 2, wherein the detectable signal emitted by the oligonucleotide if the hairpin is not formed is more intense than a signal emitted by the oligonucleotide if the hairpin is formed.

7. The method of claim 2, wherein the oligonucleotide emits the detectable signal only if the hairpin is not formed.

8. The method of claim 7, wherein the detectable signal is substantial.

9. The method of claim 2, wherein the oligonucleotide further contains a molecular energy transfer pair including an energy donor moiety that is capable of emitting energy, and an energy acceptor moiety that is capable of absorbing an amount of the emitted energy,

wherein the donor moiety is attached to a nucleotide of the second nucleotide sequence and the acceptor moiety is attached to a nucleotide of the fourth nucleotide sequence, or the acceptor moiety is attached to a nucleotide of the second nucleotide sequence and the donor moiety is attached to a nucleotide of the fourth nucleotide sequence;

and the acceptor moiety absorbs the amount of the emitted energy only if the hairpin is formed.

10. The method of claim 2, wherein the first nucleotide sequence is complementary to a nucleotide sequence flanking the target nucleotide sequence.

11. The method of claim 10, wherein the target nucleotide sequence is genomic DNA.

12. The method of claim 11, wherein the target nucleotide sequence is cDNA.

13. The method of claim 11, wherein the target nucleotide sequence is mRNA.

14. The method of claim 11, wherein the target nucleotide sequence is chemically synthesized DNA.

15. The method of claim 11, wherein the target nucleotide sequence is a sequence of an infectious disease agent.

16. The method of claim 11, wherein the target nucleotide sequence is a wild-type human genomic sequence, mutation of which is implicated in the presence of a human disease or disorder.

17. The method of claim 11, wherein the target nucleotide sequence is an amplification product.

18. The method of claim 17, wherein the amplification product contains a restriction site.

19. The method of claim 9, wherein the donor moiety is a fluorophore and the acceptor moiety is a quencher of light emitted by the fluorophore.

20. The method of claim 9, wherein the donor moiety is fluorescein.

21. The method of claim 9, wherein the donor moiety is a derivative of fluorescein.

22. The method of claim 9, wherein the donor moiety is 5-carboxyfluorescein.

23. The method of claim 9, wherein the donor moiety is rhodamine.

24. The method of claim 9, wherein the donor moiety is 5-(2'"'"'-aminoethyl) aminonapthalene-1-sulfonic acid.

25. The method of claim 9, wherein the donor moiety is anthranilamide.

26. The method of claim 9, wherein the donor moiety is coumarin.

27. The method of claim 9, wherein the donor moiety is a metal chelate.

28. The method of claim 9, wherein the donor moiety is a terbium chelate derivative.

29. The method of claim 9, wherein the donor moiety is Reactive Red 4.

30. The method of claim 9, wherein the acceptor moiety is DABCYL.

31. The method of claim 9, wherein the acceptor moiety is rhodamine.

32. The method of claim 9, wherein the acceptor moiety is pyrene butyrate.

33. The method of claim 9, wherein the acceptor moiety is eosine nitrotyrosine.

34. The method of claim 9, wherein the acceptor moiety is ethidium.

35. The method of claim 9, wherein the acceptor moiety is fluorescein.

36. The method of claim 9, wherein the acceptor moiety is a derivative of fluorescein.

37. The method of claim 9, wherein the acceptor moiety Malachite green.

38. The method of claim 9, wherein the acceptor moiety is Texas Red.

39. The method of claim 9, wherein the donor moiety is fluorescein and the acceptor moiety is DABCYL.

40. The method of claim 9, wherein the donor moiety is a derivative of fluorescein and the acceptor moiety is DABCYL.

41. The method of claim 9, wherein the nucleotide to which the donor moiety is attached is complementary to the nucleotide to which the acceptor moiety is attached.

42. The method of claim 9, wherein, if the hairpin is formed, then the nucleotide to which the donor moiety is attached and the complement of the nucleotide to which the acceptor moiety is attached are five nucleotides apart.

43. The method of claim 9, wherein the nucleotide to which the donor moiety is attached and the nucleotide to which the acceptor moiety is attached are from 15 to 25 nucleotides apart.

44. The method of claim 2 comprising, in between (b) and (c), conducting an amplification reaction, thereby incorporating the oligonucleotide into an amplification product if the target nucleotide sequence is present in the sample.

45. The method of claim 2, wherein the oligonucleotide is incorporated into the double-stranded nucleic acid using a polymerase.

46. The method of claim 44, wherein the amplification reaction is a polymerase chain reaction.

47. The method of claim 44, wherein the amplification reaction is an allele-specific polymerase chain reaction.

48. The method of claim 44, wherein the amplification reaction is a triamplification.

49. The method of claim 44, wherein the amplification reaction is a nucleic acid sequence-based amplification.

50. The method of claim 44, wherein the amplification reaction is a strand displacement amplification.

51. The method of claim 44, wherein the amplification reaction is a telomeric repeat amplification.

52. The method of claim 44, wherein the amplification reaction is a cascade rolling circle amplification.

53. The method of claim 44, wherein the amplification reaction is conducted using an amplification refractory mutation system.

54. The method of claim 44, wherein the amplification reaction is conducted in situ.

55. The method of claim 2 further comprising, prior to (b), contacting the sample with bisulfite to convert unmethylated cytosine residues to uracil residues.

56. The method of claim 2, wherein the oligonucleotide is an oligodeoxynucleotide.

57. The method of claim 2, wherein the first nucleotide sequence contains a restriction endonuclease recognition site.

58. The method of claim 2, wherein the signal is light.

59. A method for detecting a target nucleotide sequence comprising:

(a) annealing a first oligonucleotide to a nucleotide sequence flanking a target nucleotide sequence, wherein the first oligonucleotide contains;

(i) a first nucleotide sequence,

(ii) a second nucleotide sequence at the 5'"'"' end of the first nucleotide sequence,

(iii) a third nucleotide sequence at the 5'"'"' end of the second nucleotide sequence, and

(iv) a fourth nucleotide sequence at the 5'"'"' end of the third nucleotide sequence,

wherein the first oligonucleotide is capable of forming a hairpin containing nucleotides of the second and fourth nucleotide sequences, and the first oligonucleotide emits a detectable signal if the hairpin is not formed,

(b) extending the 3'"'"' end of the first oligonucleotide using the target nucleotide sequence as a template to form an extended first strand, wherein the target nucleotide sequence is annealed to the extended first strand,

(c) separating the target nucleotide sequence from the extended first strand,

(d) annealing a second oligonucleotide to the extended first strand,

(e) extending the 3'"'"' end of the second oligonucleotide using the extended first strand as a template to form an extended second strand, wherein the extended first strand is annealed to the extended second strand, and

(f) detecting the signal to detect the target nucleotide sequence.

60. The method of claim 59, wherein the first nucleotide sequence contains the nucleotide at the 3'"'"' end of the oligonucleotide, the fourth nucleotide sequence contains the nucleotide at the 5'"'"' end of the oligonucleotide, and the first nucleotide sequence is not complementary to the fourth nucleotide sequence.

61. The method of claim 60, wherein the first nucleotide sequence is not complementary to the second nucleotide sequence.

62. The method of claim 60, wherein the first nucleotide sequence is not complementary to the third nucleotide sequence.

63. The method of claim 59, wherein the detectable signal emitted by the first oligonucleotide if the hairpin is not formed is more intense than a signal emitted by the first oligonucleotide if the hairpin is formed.

64. The method of claim 59, wherein the first oligonucleotide emits the detectable signal only if the hairpin is not formed.

65. The method of claim 64, wherein the detectable signal is substantial.

66. The method of claim 59, comprising, in between (e) and (f), amplifying the extended first and second strands.

67. The method of claim 66, wherein the amplification of the extended first and second strands comprises:

(i) separating the extended first strand from the extended second strand,

(ii) annealing the first oligonucleotide to the extended second strand, and annealing the second oligonucleotide to the extended first strand,

(iii) extending the 3'"'"' end of the first oligonucleotide using the extended second strand as a template to form another extended first strand, wherein the extended second strand is annealed to the other extended first strand; and

extending the 3'"'"' end of the second oligonucleotide using the extended first strand as a template to form another extended second strand, wherein the extended first strand is annealed to the other extended second strand, and

(iv) repeating (i), (ii), and (iii) for a finite number of times, wherein, in (i), the extended first and second strands respectively are the extended first strand and the other extended second strand of (iii), or respectively are the other extended first strand and the extended second strand of (iii).

68. The method of claim 59, wherein the first oligonucleotide further contains a molecular energy transfer pair including an energy donor moiety that is capable of emitting energy, and an energy acceptor moiety that is capable of absorbing an amount of the emitted energy,

wherein the donor moiety is attached to a nucleotide of the second nucleotide sequence and the acceptor moiety is attached to a nucleotide of the fourth nucleotide sequence, or the acceptor moiety is attached to a nucleotide of the second nucleotide sequence and the donor moiety is attached to a nucleotide of the fourth nucleotide sequence;

and the acceptor moiety absorbs the amount of the emitted energy only if the hairpin is formed.

69. A method for detecting a target nucleotide sequence comprising:

(a) annealing a first oligonucleotide to a nucleotide sequence flanking a target nucleotide sequence, wherein the first oligonucleotide contains;

(i) a first nucleotide sequence complementary to the nucleotide sequence flanking the target nucleotide sequence, and

(ii) a second nucleotide sequence at the 5'"'"' end of the first nucleotide sequence,

(b) extending the 3'"'"' end of the first oligonucleotide using the target nucleotide sequence as a template to form an extended first strand, wherein the target nucleotide sequence is annealed to the extended first strand,

(c) separating the target nucleotide sequence from the extended first strand,

(d) annealing a second oligonucleotide to the extended first strand,

(e) extending the 3'"'"' end of the second oligonucleotide using the extended first strand as a template to form an extended second strand, wherein the extended first strand is annealed to the extended second strand,

(f) separating the extended first strand from the extended second strand,

(g) annealing a third oligonucleotide to the extended second strand, wherein the third oligonucleotide contains;

(i) a third nucleotide sequence,

(ii) a fourth nucleotide sequence at the 5'"'"' end of the third nucleotide sequence,

(iii) a fifth nucleotide sequence at the 5'"'"' end of the fourth nucleotide sequence, and

(iv) a sixth nucleotide sequence at the 5'"'"' end of the fifth nucleotide sequence,

wherein the third nucleotide sequence is complementary to the complement of the second nucleotide sequence, the third oligonucleotide is capable of forming a hairpin containing nucleotides of the fourth and sixth nucleotide sequences, and the third oligonucleotide emits a detectable signal if the hairpin is not formed,

(h) extending the 3'"'"' end of the third oligonucleotide using the extended second strand as a template to form a doubly extended first strand, wherein the doubly extended first strand is annealed to the extended second strand,

(i) separating the doubly extended first strand from the extended second strand,

(j) annealing the second oligonucleotide to the doubly extended first strand,

(k) extending the 3'"'"' end of the second oligonucleotide using the doubly extended first strand as a template to form a doubly extended second strand, wherein the doubly extended first strand is annealed to the doubly extended second strand, and

(l) detecting the signal to detect the target nucleotide sequence.

70. The method of claim 69, wherein the third nucleotide sequence contains the nucleotide at the 3'"'"' end of the oligonucleotide, the sixth nucleotide sequence contains the nucleotide at the 5'"'"' end of the oligonucleotide, and the third nucleotide sequence is not complementary to the sixth nucleotide sequence.

71. The method of claim 70, wherein the third nucleotide sequence is not complementary to the fourth nucleotide sequence.

72. The method of claim 70, wherein the third nucleotide sequence is not complementary to the fifth nucleotide sequence.

73. The method of claim 69, wherein the detectable signal emitted by the third oligonucleotide if the hairpin is not formed is more intense than a signal emitted by the third oligonucleotide if the hairpin is formed.

74. The method of claim 69, wherein the third oligonucleotide emits the detectable signal only if the hairpin is not formed.

75. The method of claim 74, wherein the detectable signal is substantial.

76. The method of claim 69 comprising, in between (k) and (l), amplifying the doubly extended first and second strands.

77. The method of claim 76, wherein the amplification of the doubly extended first and second strands comprises:

(i) separating the doubly extended first strand from the doubly extended second strand,

(ii) annealing the second oligonucleotide to the doubly extended first strand, and annealing the third oligonucleotide to the doubly extended second strand,

(iii) extending the 3'"'"' end of the second oligonucleotide using the doubly extended first strand as a template to form another doubly extended second strand, wherein the doubly extended first strand is annealed to the other doubly extended second strand; and

extending the 3'"'"' end of the third oligonucleotide using the doubly extended second strand as a template to form another doubly extended first strand, wherein the doubly extended second strand is annealed to the other doubly extended first strand, and

(iv) repeating (i), (ii), and (iii) for a finite number of times,

wherein, in (i), the doubly extended first and second strands respectively are the doubly extended first strand and the other doubly extended second strand of (iii), or respectively are the other doubly extended first strand and the doubly extended second strand of (iii).

78. The method of claim 69, wherein the third oligonucleotide further contains a molecular energy transfer pair including an energy donor moiety that is capable of emitting energy, and an energy acceptor moiety that is capable of absorbing an amount of the emitted energy,

wherein the donor moiety is attached to a nucleotide of the fourth nucleotide sequence and the acceptor moiety is attached to a nucleotide of the sixth nucleotide sequence, or the acceptor moiety is attached to a nucleotide of the fourth nucleotide sequence and the donor moiety is attached to a nucleotide of the sixth nucleotide sequence;

and the acceptor moiety absorbs the amount of the emitted energy only if the hairpin is formed.

79. A method for detecting a target nucleotide sequence comprising:

(a) annealing a first oligonucleotide to a nucleotide sequence flanking a target nucleotide sequence, wherein the first oligonucleotide contains;

(i) a first nucleotide sequence complementary to the nucleotide sequence flanking the target nucleotide sequence, and

(ii) a second nucleotide sequence at the 5'"'"' end of the first nucleotide sequence,

(b) extending the 3'"'"' end of the first oligonucleotide using the target nucleotide sequence as a template to form an extended first strand, wherein the target nucleotide sequence is annealed to the extended first strand,

(c) separating the target nucleotide sequence from the extended first strand,

(d) annealing a second oligonucleotide to a nucleotide sequence of the extended first strand, wherein the second oligonucleotide contains;

(i) a third nucleotide sequence complementary to the sequence of the extended first strand, and

(ii) a fourth nucleotide sequence at the 5'"'"' end of the third nucleotide sequence,

(e) extending the 3'"'"' end of the second oligonucleotide using the extended first strand as a template to form an extended second strand, wherein the extended first strand is annealed to the extended second strand,

(f) separating the extended first strand from the extended second strand,

(g) annealing a third oligonucleotide to the extended second strand, wherein the third oligonucleotide contains;

(i) a fifth nucleotide sequence,

(ii) a sixth nucleotide sequence at the 5'"'"' end of the fifth nucleotide sequence,

(iii) a seventh nucleotide sequence at the 5'"'"' end of the sixth nucleotide sequence, and

(iv) an eighth nucleotide sequence at the 5'"'"' end of the seventh nucleotide sequence,

wherein the fifth nucleotide sequence is complementary to the complement of the second nucleotide sequence, the fifth nucleotide sequence is not complementary to the eighth nucleotide sequence, the third oligonucleotide is capable of forming a first hairpin containing nucleotides of the sixth and eighth nucleotide sequences, and the third oligonucleotide emits a first detectable signal if the first hairpin is not formed,

(h) extending the 3'"'"' end of the third oligonucleotide using the extended second strand as a template to form a doubly extended first strand, wherein the doubly extended first strand is annealed to the extended second strand,

(i) separating the doubly extended first strand from the extended second strand,

(j) annealing a fourth oligonucleotide to the doubly extended first strand, wherein the fourth oligonucleotide contains;

(i) a ninth nucleotide sequence,

(ii) a tenth nucleotide sequence at the 5'"'"' end of the ninth nucleotide sequence,

(iii) an eleventh nucleotide sequence at the 5'"'"' end of the tenth nucleotide sequence, and

(iv) a twelfth nucleotide sequence at the 5'"'"' end of the eleventh nucleotide sequence,

wherein the ninth nucleotide sequence is complementary to the complement of the fourth nucleotide sequence, the ninth nucleotide sequence is not complementary to the twelfth nucleotide sequence, the fourth oligonucleotide is capable of forming a second hairpin containing nucleotides of the tenth and twelfth nucleotide sequences, and the fourth oligonucleotide emits a second detectable signal if the second hairpin is not formed,

(k) extending the 3'"'"' end of the fourth oligonucleotide using the doubly extended first strand as a template to form a doubly extended second strand, and extending the 3'"'"' end of the doubly extended first strand using the fourth oligonucleotide as a template to form a triply extended first strand, wherein the doubly extended second strand is annealed to the triply extended first strand, and

(l) detecting the first or second signal to detect the target nucleotide sequence.

80. The method of claim 79, wherein the fifth nucleotide sequence contains the nucleotide at the 3'"'"' end of the third oligonucleotide, the eighth nucleotide sequence contains the nucleotide at the 5'"'"' end of the third oligonucleotide, and the fifth nucleotide sequence is not complementary to the eighth nucleotide sequence.

81. The method of claim 80, wherein the fifth nucleotide sequence is not complementary to the sixth nucleotide sequence.

82. The method of claim 80, wherein the fifth nucleotide sequence is not complementary to the seventh nucleotide sequence.

83. The method of claim 79, wherein the ninth nucleotide sequence contains the nucleotide at the 3'"'"' end of the fourth oligonucleotide, the twelfth nucleotide sequence contains the nucleotide at the 5'"'"' end of the fourth oligonucleotide, and the ninth nucleotide sequence is not complementary to the twelfth nucleotide sequence.

84. The method of claim 83, wherein the ninth nucleotide sequence is not complementary to the tenth nucleotide sequence.

85. The method of claim 83, wherein the ninth nucleotide sequence is not complementary to the eleventh nucleotide sequence.

86. The method of claim 79, wherein the first detectable signal emitted by the third oligonucleotide if the first hairpin is not formed is more intense than a signal emitted by the third oligonucleotide if the first hairpin is formed.

87. The method of claim 79, wherein the third oligonucleotide emits the first detectable signal only if the first hairpin is not formed.

88. The method of claim 87, wherein the first detectable signal is substantial.

89. The method of claim 79, wherein the second detectable signal emitted by the fourth oligonucleotide if the second hairpin is not formed is more intense than a signal emitted by the fourth oligonucleotide if the second hairpin is formed.

90. The method of claim 79, wherein the fourth oligonucleotide emits the second detectable signal only if the second hairpin is not formed.

91. The method of claim 90, wherein the second detectable signal is substantial.

92. The method of claim 79, wherein the complete nucleotide sequences of the third and fourth oligonucleotides are identical.

93. The method of claim 79, wherein the second and fourth nucleotide sequences are identical.

94. The method of claim 79 comprising, in between (k) and (l), amplifying the doubly extended second strand and the triply extended first strand.

95. The method of claim 94, wherein the amplification of the doubly extended first and second strands comprises:

(i) separating the doubly extended second strand from the triply extended first strand,

(ii) annealing the third oligonucleotide to the doubly extended second strand, and annealing the fourth oligonucleotide to the triply extended first strand,

(iii) extending the 3'"'"' end of the third oligonucleotide using the doubly extended second strand as a template to form another triply extended first strand, wherein the doubly extended second strand is annealed to the other triply extended first strand; and

extending the 3'"'"' end of the fourth oligonucleotide using the triply extended first strand as a template to form another doubly extended second strand, wherein the triply extended first strand is annealed to the other doubly extended second strand, and

(iv) repeating (i), (ii), and (iii) for a finite number of times, wherein, in (i), the doubly extended second strand is the doubly extended second strand of (iii) and the triply extended first strand is the other triply extended first strand of (iii), or the doubly extended second strand is the other doubly extended second strand of (iii) and the triply extended first strand is the triply extended first strand of (iii).

96. The method of claim 79, wherein the third oligonucleotide further contains a molecular energy transfer pair including an energy donor moiety that is capable of emitting energy, and an energy acceptor moiety that is capable of absorbing an amount of the emitted energy,

wherein the donor moiety is attached to a nucleotide of the sixth nucleotide sequence and the acceptor moiety is attached to a nucleotide of the eighth nucleotide sequence, or the acceptor moiety is attached to a nucleotide of the sixth nucleotide sequence and the donor moiety is attached to a nucleotide of the eighth nucleotide sequence;

and the acceptor moiety absorbs the amount of the emitted energy only if the hairpin is formed.

97. The method of claim 79, wherein the fourth oligonucleotide further contains a molecular energy transfer pair including an energy donor moiety that is capable of emitting energy, and an energy acceptor moiety that is capable of absorbing an amount of the emitted energy,

wherein the donor moiety is attached to a nucleotide of the tenth nucleotide sequence and the acceptor moiety is attached to a nucleotide of the twelfth nucleotide sequence, or the acceptor moiety is attached to a nucleotide of the tenth nucleotide sequence and the donor moiety is attached to a nucleotide of the twelfth nucleotide sequence;

and the acceptor moiety absorbs the amount of the emitted energy only if the hairpin is formed.

98. A method for determining if a target nucleotide sequence is present in a sample comprising:

(a) contacting the sample with first and second oligonucleotides, wherein the first oligonucleotide is capable of annealing to the second oligonucleotide to form a duplex, and the first or second oligonucleotide emits a detectable signal if the duplex is not formed,

(b) incorporating the first or second oligonucleotide into a double-stranded nucleic acid using a polymerase if the target nucleotide sequence is present in the sample, thereby preventing the first and second nucleotides from forming the duplex, and

(c) determining that the target nucleotide sequence is present in the sample if the signal is detected, or determining that the target nucleotide sequence is not present in the sample if the signal is not detected.

99. The method of claim 98, wherein the detectable signal emitted by the first or second oligonucleotide if the first oligonucleotide is not annealed to the second oligonucleotide is more intense than a signal emitted by the first or second oligonucleotide if the first oligonucleotide is annealed to the second oligonucleotide.

100. The method of claim 98, wherein the first or second oligonucleotide emits the detectable signal only if the first oligonucleotide is not annealed to the second oligonucleotide.

101. The method of claim 100, wherein the detectable signal is substantial.

102. The method of claim 98, wherein the first and second oligonucleotides contain a molecular energy transfer pair including an energy donor moiety that is capable of emitting energy, and an energy acceptor moiety that is capable of absorbing an amount of the emitted energy,

wherein the donor moiety is attached to a nucleotide of the first oligonucleotide and the acceptor moiety is attached to a nucleotide of the second oligonucleotide, or the acceptor moiety is attached to a nucleotide of the first oligonucleotide and the donor moiety is attached to a nucleotide of the second oligonucleotide, and the acceptor moiety absorbs the amount of energy only if the duplex is formed.

103. The method of claim 98 comprising, in between (b) and (c), conducting an amplification reaction, thereby incorporating the first or second oligonucleotide into an amplification product if the target nucleotide sequence is present in the sample.