CIRCULAR PROBE AMPLIFICATION (CPA) USING ENERGY-TRANSFER PRIMERS
First Claim
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1. A nucleic acid amplification method comprising:
- (a) providing a closed circular padlock probe molecule;
a target nucleic acid molecule;
a forward primer;
a reverse primer;
dNTPs; and
a first DNA polymerase to form a reaction mixture;
(b) creating a multi-tailed complex;
(c) activating a second DNA polymerase, wherein said second DNA polymerase is thermostable; and
(d) thermocycling said multi-tailed complex.
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Abstract
The present invention provides methods and kits for the rapid exponential amplification of nucleic acid molecules using a padlock probe. The present invention improves upon the existing methods for amplifying padlock probes by eliminating or delaying the appearance of artifact products that cause false positive results, and also increase the sensitivity and speed of the assay. Further provided are nucleic acid amplification primers containing non-informative base analogs.
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Citations
61 Claims
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1. A nucleic acid amplification method comprising:
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(a) providing a closed circular padlock probe molecule;
a target nucleic acid molecule;
a forward primer;
a reverse primer;
dNTPs; and
a first DNA polymerase to form a reaction mixture;
(b) creating a multi-tailed complex;
(c) activating a second DNA polymerase, wherein said second DNA polymerase is thermostable; and
(d) thermocycling said multi-tailed complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A nucleic acid molecule amplification kit comprising:
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(a) a forward primer and a reverse primer;
(b) a ligase enzyme;
(c) a first polymerase enzyme;
(d) a linear padlock probe molecule, wherein the padlock probe comprises a 3′
terminal region, a 5′
terminal region, and a spacer region, wherein the spacer region contains binding sites for the forward primer and the reverse primer; and
(e) a second polymerase enzyme, wherein the second polymerase enzyme is a thermostable enzyme and wherein the second polymerase enzyme is not the same enzyme as the first polymerase enzyme. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method for detecting a target nucleic acid molecule in a sample comprising:
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(a) providing a target nucleic acid molecule, a linear padlock probe molecule, a ligase enzyme, a forward primer, a reverse primer, dNTPs, and a first DNA polymerase;
(b) creating a closed circular padlock probe molecule;
(c) creating a multi-tailed complex from said closed circular padlock probe molecule;
(d) activating a second DNA polymerase;
(e) thermocycling said multi-tailed complex with said second DNA polymerase; and
(f) detecting the amplification product of said multi-tailed complex.
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34. A method for detecting a target nucleic acid molecule in a sample comprising:
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(a) providing a target nucleic acid molecule, a closed circular padlock probe molecule topologically linked to said target nucleic acid molecule, a forward primer, a reverse primer, dNTPs, and a first DNA polymerase;
(b) creating a multi-tailed complex from said closed circular padlock probe molecule;
(c) activating a second DNA polymerase;
(d) thermocycling said multi-tailed complex with said second DNA polymerase; and
(e) detecting the amplification product of said multi-tailed complex.
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35. A method for detecting a plurality of target nucleic acid molecules in a sample comprising:
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(a) providing a plurality of target nucleic acid molecules, a plurality of linear padlock probe molecules capable of annealing to a plurality of distinct target nucleic acid molecule, a ligase enzyme, dNTPs, and a first DNA polymerase;
(b) creating at least two closed circular nucleic acid molecules, wherein each of said closed circular nucleic acid molecules is topologically linked to a distinct target nucleic acid molecule;
(c) providing, for each member of said at least two closed circular nucleic acid molecules, a forward primer and a reverse primer;
(d) creating a multi-tailed complex for each of said distinct target nucleic acid molecules;
(e) activating a second DNA polymerase;
(f) thermocycling said at least two distinct multi-tailed complexes with said second DNA polymerase; and
(g) detecting the amplification products of said at least two distinct multi-tailed complexes. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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53. A closed tube nucleic acid molecule amplification method comprising:
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(a) providing a target nucleic acid molecule, a ligase enzyme;
a forward primer;
a reverse primer;
dNTPs; and
a first DNA polymerase in a reaction tube;
(b) sealing said reaction tube;
(c) creating a closed circular padlock probe molecule;
(d) creating a multi-tailed complex from said closed circular padlock probe molecule;
(e) activating a second DNA polymerase, wherein said second DNA polymerase is a thermostable DNA polymerase; and
(f) thermocycling said multi-tailed complex. - View Dependent Claims (54, 55)
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- 56. A padlock probe amplification primer comprising any of the following sequences:
- 59. A padlock probe amplification primer of 15 to 75 nucleotides wherein at least one nucleotide base contained in the primer is a non-informative base analog.
Specification