Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon
First Claim
1. An oligodeoxynucleotide, the sequence of which consists of:
- 5'"'"'-GGCTACGAACCAGGTAAGCCGTA-3'"'"' (SEQ ID NO;
1), wherein fluorescein or a derivative thereof is attached to the 5'"'"' G and DABCYL is attached to the T at nucleotide number 22.
18 Assignments
0 Petitions
Accused Products
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.
251 Citations
104 Claims
-
1. An oligodeoxynucleotide, the sequence of which consists of:
- 5'"'"'-GGCTACGAACCAGGTAAGCCGTA-3'"'"' (SEQ ID NO;
1), wherein fluorescein or a derivative thereof is attached to the 5'"'"' G and DABCYL is attached to the T at nucleotide number 22.
- 5'"'"'-GGCTACGAACCAGGTAAGCCGTA-3'"'"' (SEQ ID NO;
-
2. An oligodeoxynucleotide, the sequence of which consists of:
- 5'"'"'-ACCTTCTGTTCACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
14), wherein fluorescein or a derivative thereof is attached to the 5'"'"' A and DABCYL is attached to the T at nucleotide number 24.
- 5'"'"'-ACCTTCTGTTCACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
-
3. An oligodeoxynucleotide, the sequence of which consists of:
- 5'"'"'-ACCTTCGATTCACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
15), wherein fluorescein or a derivative thereof is attached to the 5'"'"' A and DABCYL is attached to the T at nucleotide number 24.
- 5'"'"'-ACCTTCGATTCACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
-
4. An oligodeoxynucleotide, the sequence of which consists of:
- 5'"'"'-ACCTTCTGTACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
16), wherein fluorescein or a derivative thereof is attached to the 5'"'"' A and DABCYL is attached to the T at nucleotide number 22.
- 5'"'"'-ACCTTCTGTACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
-
5. An oligodeoxynucleotide, the sequence of which consists of:
- 5'"'"'-ACCTTCTATACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
17), wherein fluorescein or a derivative thereof is attached to the 5'"'"' A and DABCYL is attached to the T at nucleotide number 22.
- 5'"'"'-ACCTTCTATACCCTCAGAAGGTGACCAAGTTCAT-3'"'"' (SEQ ID NO;
-
6. An oligonucleotide containing:
-
(a) a first nucleotide sequence, (b) a second nucleotide sequence at the 5'"'"' end of the first nucleotide sequence, (c) a third nucleotide sequence at the 5'"'"' end of the second nucleotide sequence, and (d) a fourth nucleotide sequence at the 5'"'"' end of the third nucleoide sequence, wherein the first nucleotide sequence contains the nucleotide at the 3'"'"' end of the oligonucleotide and the fourth nucleotide sequence contains the nucleotide at the 5'"'"' end of the oligonucleotide, wherein a plurality of nucleotides of the first nucleotide sequence is unable to form part of a hairpin capable of being formed by the oligonucleotide, wherein, if the hairpin is formed, then the hairpin contains nucleotides of the second and fourth nucleotide sequences, and wherein, if the hairpin is not formed, then the oligonucleotide emits a detectable signal.
-
-
7. The oligonucleotide of claim 6, wherein the first nucleotide sequence is not complementary to the second nucleotide sequence.
-
8. The oligonucleotide of claim 6, wherein the first nucleotide sequence is not complementary to the third nucleotide sequence.
-
9. The oligonucleotide of claim 6, 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.
-
10. The oligonucleotide of claim 6, wherein the oligonucleotide emits the detectable signal only if the hairpin is not formed.
-
11. The oligonucleotide of claim 10, wherein the detectable signal is substantial.
-
12. The oligonucleotide of claim 6 further containing 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.
-
13. The oligonucleotide of claim 6, wherein the first nucleotide sequence is complementary to a nucleotide sequence flanking a target nucleotide sequence.
-
14. The oligonucleotide of claim 13, wherein the target nucleotide sequence is genomic DNA.
-
15. The oligonucleotide of claim 13, wherein the target nucleotide sequence is cDNA.
-
16. The oligonucleotide of claim 13, wherein the target nucleotide sequence is mRNA.
-
17. The oligonucleotide of claim 13, wherein the target nucleotide sequence is chemically synthesized DNA.
-
18. The oligonucleotide of claim 13, wherein the target nucleotide sequence is a sequence of an infectious disease agent.
-
19. The oligonucleotide of claim 13, 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.
-
20. The method of claim 13, wherein the target nucleotide sequence is an amplification product.
-
21. The method of claim 20, wherein the amplification product contains a restriction endonuclease recognition site.
-
22. The oligonucleotide of claim 12, wherein the donor moiety is a fluorophore and the acceptor moiety is a quencher of light emitted by the fluorophore.
-
23. The oligonucleotide of claim 12, wherein the donor moiety is fluorescein.
-
24. The oligonucleotide of claim 12, wherein the donor moiety is a derivative of fluorescein.
-
25. The oligonucleotide of claim 12, wherein the donor moiety is 5-carboxyfluorescein.
-
26. The oligonucleotide of claim 12, wherein the donor moiety is rhodamine.
-
27. The oligonucleotide of claim 12, wherein the donor moiety is 5-(2'"'"'-aminoethyl) aminonapthalene-1-sulfonic acid.
-
28. The oligonucleotide of claim 12, wherein the donor moiety is anthranilamide.
-
29. The oligonucleotide of claim 12, wherein the donor moiety is coumarin.
-
30. The oligonucleotide of claim 12, wherein the donor moiety is a metal chelate.
-
31. The oligonucleotide of claim 12, wherein the donor moiety is a terbium chelate derivative.
-
32. The oligonucleotide of claim 12, wherein the donor moiety is Reactive Red 4.
-
33. The oligonucleotide of claim 12, wherein the acceptor moiety is DABCYL.
-
34. The oligonucleotide of claim 12, wherein the acceptor moiety is rhodamine.
-
35. The oligonucleotide of claim 12, wherein the acceptor moiety is pyrene butyrate.
-
36. The oligonucleotide of claim 12, wherein the acceptor moiety is eosine nitrotyrosine.
-
37. The oligonucleotide of claim 12, wherein the acceptor moiety is ethidium.
-
38. The oligonucleotide of claim 12, wherein the acceptor moiety is fluorescein.
-
39. The oligonucleotide of claim 12, wherein the acceptor moiety is a derivative of fluorescein.
-
40. The oligonucleotide of claim 12, wherein the acceptor moiety is Malachite green.
-
41. The oligonucleotide of claim 12, wherein the acceptor moiety is Texas Red.
-
42. The oligonucleotide of claim 12, wherein the donor moiety is fluorescein and the acceptor moiety is DABCYL.
-
43. The oligonucleotide of claim 12, wherein the donor moiety is a derivative of fluorescein and the acceptor moiety is DABCYL.
-
44. The oligonucleotide of claim 12, wherein the nucleotide to which the donor moiety is attached is complementary to the nucleotide to which the acceptor moiety is attached.
-
45. The oligonucleotide of claim 12, 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.
-
46. The oligonucleotide of claim 12, 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.
-
47. The oligonucleotide of claim 6, wherein the oligonucleotide is an oligodeoxynucleotide.
-
48. The oligonucleotide of claim 6, wherein the first nucleotide sequence contains a restriction endonuclease recognition site.
-
49. The oligonucleotide of claim 6, wherein the signal is light.
-
50. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 1, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
1, and DABCYL is attached to the nucleotide at position 22 of SEQ ID NO;
1.
- 1, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
51. The oligonucleotide of claim 50 consisting of the nucleotide sequence of SEQ ID NO:
- 1.
-
52. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 1, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
1, and DABCYL is attached to the nucleotide at position 22 of SEQ ID NO;
1.
- 1, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
53. The oligonucleotide of claim 6 consisting of the nucleotide sequence of SEQ ID NO:
- 1.
-
54. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 13, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
13, and DABCYL is attached to the nucleotide at position 20 of SEQ ID NO;
13.
- 13, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
55. The oligonucleotide of claim 54 consisting of the nucleotide sequence of SEQ ID NO:
- 13.
-
56. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 13, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
13, and DABCYL is attached to the nucleotide at position 20 of SEQ ID NO;
13.
- 13, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
57. The oligonucleotide of claim 56 consisting of the nucleotide sequence of SEQ ID NO:
- 13.
-
58. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 14, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
14, and DABCYL is attached to the nucleotide at position 24 of SEQ ID NO;
14.
- 14, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
59. The oligonucleotide of claim 58 consisting of the nucleotide sequence of SEQ ID NO:
- 14.
-
60. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 14, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
14, and DABCYL is attached to the nucleotide at position 24 of SEQ ID NO;
14.
- 14, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
61. The oligonucleotide of claim 60 consisting of the nucleotide sequence of SEQ ID NO:
- 14.
-
62. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 15, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
15, and DABCYL is attached to the nucleotide at position 24 of SEQ ID NO;
15.
- 15, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
63. The oligonucleotide of claim 62 consisting of the nucleotide sequence of SEQ ID NO:
- 15.
-
64. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 15, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
15, and DABCYL is attached to the nucleotide at position 24 of SEQ ID NO;
15.
- 15, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
65. The oligonucleotide of claim 64 consisting of the nucleotide sequence of SEQ ID NO:
- 15.
-
66. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 16, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
16, and DABCYL is attached to the nucleotide at position 22 of SEQ ID NO;
16.
- 16, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
67. The oligonucleotide of claim 66 consisting of the nucleotide sequence of SEQ ID NO:
- 16.
-
68. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 16, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
16, and DABCYL is attached to the nucleotide at position 22 of SEQ ID NO;
16.
- 16, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
69. The oligonucleotide of claim 68 consisting of the nucleotide sequence of SEQ ID NO:
- 16.
-
70. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 17, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
17, and DABCYL is attached to the nucleotide at position 22 of SEQ ID NO;
17.
- 17, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
71. The oligonucleotide of claim 70 consisting of the nucleotide sequence of SEQ ID NO:
- 17.
-
72. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 17, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
17, and DABCYL is attached to the nucleotide at position 22 of SEQ ID NO;
17.
- 17, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
73. The oligonucleotide of claim 72 consisting of the nucleotide sequence of SEQ ID NO:
- 17.
-
74. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 18, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
18, and DABCYL is attached to the nucleotide at position 20 of SEQ ID NO;
18.
- 18, wherein fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
75. The oligonucleotide of claim 74 consisting of the nucleotide sequence of SEQ ID NO:
- 18.
-
76. An oligonucleotide comprising the nucleotide sequence of SEQ ID NO:
- 18, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
18, and DABCYL is attached to the nucleotide at position 20 of SEQ ID NO;
18.
- 18, wherein a derivative of fluorescein is attached to the nucleotide at position 1 of SEQ ID NO;
-
77. The oligonucleotide of claim 76 consisting of the nucleotide sequence of SEQ ID NO:
- 18.
-
78. A kit comprising:
-
(a) a polymerase, and (b) 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.
-
-
79. The kit of claim 78, 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.
-
80. The kit of claim 79, wherein the first nucleotide sequence is not complementary to the second nucleotide sequence.
-
81. The kit of claim 79, wherein the first nucleotide sequence is not complementary to the third nucleotide sequence.
-
82. The kit of claim 78, 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.
-
83. The kit of claim 78, wherein the oligonucleotide emits the detectable signal only if the hairpin is not formed.
-
84. The kit of claim 83, wherein the detectable signal is substantial.
-
85. The kit of claim 78, 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.
-
86. The kit of claim 78, wherein the polymerase is a DNA polymerase.
-
87. The kit of claim 78, wherein the oligonucleotide is an oligodeoxynucleotide.
-
88. The kit of claim 78 further comprising a ligase in a container that does not contain the polymerase or the oligonucleotide.
-
89. A kit comprising first and second oligonucleotides,
wherein the first oligonucleotide contains: -
(i) a first nucleotide sequence complementary to a nucleotide sequence flanking a target nucleotide sequence, and (ii) a second nucleotide sequence at the 5" end of the first nucleotide sequence, and wherein the second 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 second oligonucleotide is capable of forming a hairpin containing nucleotides of the fourth and sixth nucleotide sequences, and the second oligonucleotide emits a detectable signal if the hairpin is not formed.
-
-
90. The kit of claim 89, 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.
-
91. The kit of claim 90, wherein the third nucleotide sequence is not complementary to the fourth nucleotide sequence.
-
92. The kit of claim 90, wherein the third nucleotide sequence is not complementary to the fifth nucleotide sequence.
-
93. The kit of claim 89, wherein the second 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.
-
94. The kit of claim 89 further comprising a polymerase.
-
95. The kit of claim 94, wherein the polymerase is a DNA polymerase.
-
96. The kit of claim 89, wherein the first oligonucleotide is an oligodeoxynucleotide.
-
97. The kit of claim 89, wherein the second oligonucleotide is an oligodeoxynucleotide.
-
98. A kit comprising:
-
(a) a polymerase, (b) a first oligonucleotide, and (c) a second oligonucleotide, wherein the first oligonucleotide is capable of annealing to the second oligonucleotide, and the first or second oligonucleotide emits a detectable signal only if the first oligonucleotide is not annealed to the second oligonucleotide.
-
-
99. The kit 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 kit 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 kit of claim 100, wherein the detectable signal is substantial.
-
102. The kit of claim 98 further comprising a molecular energy 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 first oligonucleotide is annealed to the second oligonucleotide.
-
103. The oligonucleotide of claim 6, wherein the first nucleotide sequence is not complementary to the fourth nucleotide sequence.
-
104. The oligonucleotide of claim 6, wherein the oligonucleotide is capable of forming only one hairpin.
Specification