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) incubating said reaction mixture at about 50°
C. to about 70°
C. for 1 minute to 30 minutes;
(c) creating a multi-tailed complex by cascade rolling circle amplification wherein the multi-tailed complex has multiple primer binding sites, and wherein the multi-tailed complex is not a detectable amplification product;
(d) activating a second DNA polymerase, wherein said second DNA polymerase is thermostable; and
(e) thermocycling said multi-tailed complex with said second DNA polymerase, wherein said thermocycling said multi-tailed complex generates a detectable amplification product.
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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
41 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) incubating said reaction mixture at about 50°
C. to about 70°
C. for 1 minute to 30 minutes;(c) creating a multi-tailed complex by cascade rolling circle amplification wherein the multi-tailed complex has multiple primer binding sites, and wherein the multi-tailed complex is not a detectable amplification product; (d) activating a second DNA polymerase, wherein said second DNA polymerase is thermostable; and (e) thermocycling said multi-tailed complex with said second DNA polymerase, wherein said thermocycling said multi-tailed complex generates a detectable amplification product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 29, 30, 31, 32, 33, 34, 35)
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13. 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) incubating at about 50°
C. to about 70°
C. for 1 minute to 30 minutes;(d) creating a multi-tailed complex from said closed circular padlock probe molecule by cascade rolling circle amplification wherein the multi-tailed complex has multiple primer binding sites, and wherein the multi-tailed complex is not a detectable amplification product; (e) activating a second DNA polymerase; (f) thermocycling said multi-tailed complex with said second DNA polymerase wherein said thermocycling said multi-tailed complex generates a detectable amplification product; and (g) detecting the amplification product of said multi-tailed complex.
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14. 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) incubating at about 50°
C. to about 70°
C. for 1 minute to 30 minutes;(c) creating a multi-tailed complex from said closed circular padlock probe molecule by cascade rolling circle amplification wherein the multi-tailed complex has multiple primer binding sites, and wherein the multi-tailed complex is not a detectable amplification product; (d) activating a second DNA polymerase; (e) thermocycling said multi-tailed complex with said second DNA polymerase wherein said thermocycling said multi-tailed complex generates a detectable amplification product; and (f) detecting the amplification product of said multi-tailed complex.
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15. 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) incubating at about 50°
C. to about 70°
C. for 1 minute to 30 minutes;(e) creating a multi-tailed complex for each of said distinct target nucleic acid molecules by cascade rolling circle amplification wherein the multi-tailed complex has multiple primer binding sites, and wherein the multi-tailed complex is not a detectable amplification product; (f) activating a second DNA polymerase; (g) thermocycling said at least two distinct multi tailed complexes with said second DNA polymerase wherein said thermocycling said at least two distinct multi-tailed complexes generates detectable amplification products; and (h) detecting the amplification products of said at least two distinct multi-tailed complexes. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 39, 40, 41)
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26. 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) incubating at about 50°
C. to about 70°
C. for 1 minute to 30 minutes;(e) creating a multi-tailed complex from said closed circular padlock probe molecule by cascade rolling circle amplification wherein the multi-tailed complex has multiple primer binding sites, and wherein the multi-tailed complex is not a detectable amplification product; (f) activating a second DNA polymerase, wherein said second DNA polymerase is a thermostable DNA polymerase; and (g) thermocycling said multi-tailed complex with said second DNA polymerase, wherein said thermocycling said multi-tailed complex generates a detectable amplification product. - View Dependent Claims (27, 28)
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Specification