Thermophilic Helicase Dependent Amplification Technology With Endpoint Homogenous Fluorescent Detection
First Claim
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1. A method of amplifying a double stranded target nucleic acid in a helicase-dependent reaction, comprising:
- (a) denaturing the target nucleic acid;
(b) contacting one or more oligonucleotide probes with the denatured target nucleic acid, wherein one or more of the oligonucleotide probes hybridize to the denatured target nucleic acid to form double-stranded probe-target hybrids;
(c) contacting the double-stranded probe-target hybrids with one or more capture antibodies wherein the one or more capture antibodies bind to the double-stranded probe-target hybrids to form captured double-stranded probe-target hybrids,(d) removing all uncaptured nucleic acids;
(e) adding one or more oligonucleotide primers, wherein the oligonucleotide primers hybridize to the target nucleic acid;
(f) synthesizing an extension product of the oligonucleotide primers which is complementary to the target nucleic acid, by means of a DNA polymerase to form a target nucleic acid duplex;
(g) contacting the target nucleic acid duplex of step (f) with a helicase preparation and amplifying the target nucleic acid duplex in a helicase-dependent reaction.
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Abstract
Disclosed herein are methods of amplifying a target nucleic acid in a helicase-dependent reaction. Also disclosed are methods of amplifying and detecting a target nucleic acid in a helicase-dependent reaction as well as modified detection labels to assist in the detection.
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Citations
79 Claims
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1. A method of amplifying a double stranded target nucleic acid in a helicase-dependent reaction, comprising:
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(a) denaturing the target nucleic acid; (b) contacting one or more oligonucleotide probes with the denatured target nucleic acid, wherein one or more of the oligonucleotide probes hybridize to the denatured target nucleic acid to form double-stranded probe-target hybrids; (c) contacting the double-stranded probe-target hybrids with one or more capture antibodies wherein the one or more capture antibodies bind to the double-stranded probe-target hybrids to form captured double-stranded probe-target hybrids, (d) removing all uncaptured nucleic acids; (e) adding one or more oligonucleotide primers, wherein the oligonucleotide primers hybridize to the target nucleic acid; (f) synthesizing an extension product of the oligonucleotide primers which is complementary to the target nucleic acid, by means of a DNA polymerase to form a target nucleic acid duplex; (g) contacting the target nucleic acid duplex of step (f) with a helicase preparation and amplifying the target nucleic acid duplex in a helicase-dependent reaction. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A method of amplifying a single stranded target nucleic acid in a helicase-dependent reaction, comprising:
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(a) contacting one or more oligonucleotide probes with the single stranded target nucleic acid, wherein one or more of the oligonucleotide probes hybridize to the target nucleic acid to form double-stranded probe-target hybrids; (b) contacting the double-stranded probe-target hybrids with one or more capture antibodies, wherein the one or more of capture antibodies bind to the double-stranded probe-target hybrids to form captured double-stranded probe-target hybrids, (c) removing all uncaptured nucleic acids; (d) adding one or more oligonucleotide primers, wherein the oligonucleotide primers hybridize to the target nucleic acid; (e) synthesizing an extension product of the oligonucleotide primers which is complementary to the target nucleic acid, by means of a DNA polymerase to form a target nucleic acid duplex; (f) contacting the target nucleic acid duplex of step (e) with a helicase preparation and amplifying the target nucleic acid duplex in a helicase-dependent reaction. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78)
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79. A method of amplifying two double stranded target nucleic acids in a single helicase-dependent reaction, wherein the two double stranded target nucleic acids comprise a first and a second double stranded target nucleic acids comprising:
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(a) denaturing the target nucleic acids; (b) contacting the first denatured target nucleic acid with one or more oligonucleotide probes wherein the oligonucleotide probes hybridize to the first denatured target nucleic acid to form first target double-stranded probe-target hybrids, and contacting the second denatured target nucleic acid with one or more oligonucleotide probes wherein the oligonucleotide probes hybridize to the second denatured target nucleic acid to form second target double-stranded probe-target hybrids; (c) contacting the first and second double-stranded probe-target hybrids with one or more capture antibodies, wherein the one or more capture antibodies bind to the first and second double-stranded probe-target hybrids to form captured first and second double-stranded probe-target hybrids, (d) removing all uncaptured nucleic acids; (e) adding one or more first target oligonucleotide primers, wherein the first target oligonucleotide primers hybridize to the first target nucleic acid and adding one or more second target oligonucleotide primers, wherein the second target oligonucleotide primers hybridize to the second target nucleic acid; (f) synthesizing extension products of the first and second target oligonucleotide primers which are complementary to the first and second target nucleic acids, respectively, by means of a DNA polymerase to form first and second target nucleic acid duplexes; (g) contacting the first and second target nucleic acid duplexes of step (f) with a helicase preparation and amplifying the target nucleic acid duplexes in a helicase-dependent reaction, wherein the helicase preparation comprises one or more primers that hybridize to the first target nucleic acid and further comprises one or more primers that hybridize to the second target nucleic acid.
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Specification