Probes for specific analysis of nucleic acids
First Claim
1. A method for detecting or enriching for a target deoxyribonucleic acid (DNA) present in a nucleic acid sample, said method comprising:
- (a) fragmenting a nucleic acid sample to generate nucleic acid fragments including a target fragment containing said target DNA;
(b) non-specifically ligating an adaptor sequence to an end of said fragments, wherein the adaptor sequence becomes ligated to an end of a strand of said fragments and wherein the end of said fragments to which said adaptor sequence is ligated is the other end of the same strand to which oligonucleotide B of the probe is ligated in step (e);
(c) rendering said fragments, including said target fragment, at least partially single-stranded, wherein the single-stranded portion includes an end portion and wherein the length of said single-stranded portion is sufficient to allow hybridisation of at least part of the single-stranded portion of said target fragment to the probe of step (d), wherein said rendering is done by denaturation or using an exonuclease;
(d) contacting the at least partially single-stranded fragments of step (c) with oligonucleotides A and B of a single target-specific nucleic acid probe, wherein;
(i) oligonucleotide A is a single-stranded oligonucleotide comprising at one end a first target-specific part comprising at least 10 nucleotides complementary in sequence to at least part of said single-stranded portion of said target fragment, and comprising at the other end a second non-target-specific part which comprises a nucleotide sequence complementary to at least a portion, including one end, of oligonucleotide B of the probe, and(ii) oligonucleotide B is a single-stranded oligonucleotide which may contain or carry at least one element for detection and/or enrichment of said target fragment, and of which at least a portion, including one end, is complementary in sequence to the second non-target-specific part of oligonucleotide A,such that said target fragment becomes annealed to said probe through hybridisation to the first target-specific part of oligonucleotide A resulting in only one target-specific probe-binding event per target fragment;
(e) directly or indirectly ligating oligonucleotide B of said probe to the part of the single-stranded portion of said target fragment which is hybridised to oligonucleotide A of said probe to produce a probe-target fragment hybrid; and
(f) detecting or enriching for said probe-target fragment hybrid.
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Abstract
The present invention provides a method for detecting or enriching for a target deoxyribonucleic acid (DNA) present in a nucleic acid sample, said method comprising: (a) fragmenting a nucleic acid sample to generate nucleic acid fragments including a target fragment containing said target DNA and non-specifically ligating an adaptor sequence to an end of said fragments; (b) rendering said fragments at least partially single-stranded; (c) contacting the at least partially single-stranded fragments of step (b) with oligonucleotides A and B of a single target-specific nucleic acid probe; (d) ligating oligonucleotide B of said probe to the part of the single-stranded portion of said target fragment which is hybridised to oligonucleotide A of said probe to produce a probe-target fragment hybrid; and (e) detecting or enriching for said probe-target fragment hybrid.
52 Citations
18 Claims
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1. A method for detecting or enriching for a target deoxyribonucleic acid (DNA) present in a nucleic acid sample, said method comprising:
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(a) fragmenting a nucleic acid sample to generate nucleic acid fragments including a target fragment containing said target DNA; (b) non-specifically ligating an adaptor sequence to an end of said fragments, wherein the adaptor sequence becomes ligated to an end of a strand of said fragments and wherein the end of said fragments to which said adaptor sequence is ligated is the other end of the same strand to which oligonucleotide B of the probe is ligated in step (e); (c) rendering said fragments, including said target fragment, at least partially single-stranded, wherein the single-stranded portion includes an end portion and wherein the length of said single-stranded portion is sufficient to allow hybridisation of at least part of the single-stranded portion of said target fragment to the probe of step (d), wherein said rendering is done by denaturation or using an exonuclease; (d) contacting the at least partially single-stranded fragments of step (c) with oligonucleotides A and B of a single target-specific nucleic acid probe, wherein; (i) oligonucleotide A is a single-stranded oligonucleotide comprising at one end a first target-specific part comprising at least 10 nucleotides complementary in sequence to at least part of said single-stranded portion of said target fragment, and comprising at the other end a second non-target-specific part which comprises a nucleotide sequence complementary to at least a portion, including one end, of oligonucleotide B of the probe, and (ii) oligonucleotide B is a single-stranded oligonucleotide which may contain or carry at least one element for detection and/or enrichment of said target fragment, and of which at least a portion, including one end, is complementary in sequence to the second non-target-specific part of oligonucleotide A, such that said target fragment becomes annealed to said probe through hybridisation to the first target-specific part of oligonucleotide A resulting in only one target-specific probe-binding event per target fragment; (e) directly or indirectly ligating oligonucleotide B of said probe to the part of the single-stranded portion of said target fragment which is hybridised to oligonucleotide A of said probe to produce a probe-target fragment hybrid; and (f) detecting or enriching for said probe-target fragment hybrid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification