Nucleic acid sequence detection employing probes comprising non-nucleosidic coumarin derivatives as polynucleotide-crosslinking agents
First Claim
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1. A method of detecting a target nucleic acid in a sample, said method comprising the steps of:
- a) hybridizing a target nucleic acid to a crosslinkable probe, said crosslinkable probe comprising;
i) a polynucleotide having a sequence complementary to that of said target nucleic acid;
ii) a crosslinking moiety comprising a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumain derivative is linked to a (poly)hydroxy hydrocarbon backbone moiety other than ribose or deoxyribose; and
iii) optionally at least one label;
b) activating said crosslinking moiety, whereby a covalent crosslink occurs between said probe and said target nucleic acid; and
c) detecting the presence of a crosslinked nucleic acid pair as indicative of the presence of said target sequence in said sample.
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Abstract
Methods and compositions are provided for detecting nucleic acid sequences. Probes comprising a crosslinking agent are combined with a sample which may comprise a target sequence which is complementary to the probe. Hybridization is allowed to occur between complementary sequences. The crosslinking agent is activated. Covalent bonds are formed between the probe and the target sequence if they are hybridized to one another. The crosslinked nucleic acids can then be detected to indicate the presence of the target sequence. Also provided are kits comprising reagents.
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Citations
24 Claims
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1. A method of detecting a target nucleic acid in a sample, said method comprising the steps of:
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a) hybridizing a target nucleic acid to a crosslinkable probe, said crosslinkable probe comprising;
i) a polynucleotide having a sequence complementary to that of said target nucleic acid;
ii) a crosslinking moiety comprising a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumain derivative is linked to a (poly)hydroxy hydrocarbon backbone moiety other than ribose or deoxyribose; and
iii) optionally at least one label;
b) activating said crosslinking moiety, whereby a covalent crosslink occurs between said probe and said target nucleic acid; and
c) detecting the presence of a crosslinked nucleic acid pair as indicative of the presence of said target sequence in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
d) isolating said crosslinked nucleic acid pair.
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11. The method of claim 1 wherein said target nucleic acid in a sample is genomic DNA.
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12. The method of claim 1 wherein said target nucleic acid in a sample is genomic DNA derived from whole blood.
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13. A method according to claim 1 wherein multiple samples are assayed for one or more target nucleic acids in an automated system.
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14. A kit including components for detecting a target nucleic acid in a sample, said kit comprising:
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a) a crosslinkable probe comprising;
i) a polynucleotide having a sequence complementary to that of said target and capable of hybridizing with said target nucleic acid;
ii) a crosslinking moiety comprising a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative is linked to a (poly)hydroxy hydrocarbon backbone moiety other than ribose or deoxyribose; and
iii) optionally at least one label; and
b) a control. - View Dependent Claims (15, 16, 17, 18)
i) a polynucleotide having a sequence complementary to that of said target nucleic acid;
ii) a crosslinking moiety comprising a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative is linked to a (poly)hydroxy hydrocarbon backbone moiety other than ribose or deoxyribose; and
iii) optionally at least one label.
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16. A kit according to claim 15 wherein each of said first and second crosslinkable probes are characterized by having a label, wherein one of said labels is a member of a specific binding pair, and one of said labels provides a detectable signal.
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17. The kit of claim 14 wherein said control is a purified plasmid containing the nucleic acid sequence to be detected.
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18. The kit of claim 14 wherein said control is an amplicon of the region of the target nucleic acid to be detected.
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19. A method of detecting a target nucleic acid in a sample, said method comprising the steps of:
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a) hybridizing a target nucleic acid to at least a first and a second crosslinkable probe, said crosslinkable probes comprising;
i) a polynucleotide having a sequence complementary to that of said target nucleic acid;
ii) a crosslinking moiety comprising a non-nucleosidic coumarin derivative incorporated into the backbone of said polynucleotide such that it replaces one or more nucleotides otherwise complementary to said target nucleic acid, wherein said coumarin derivative is linked to a (poly)hydroxy hydrocarbon backbone moiety other than ribose or deoxyribose; and
iii) optionally at least one label;
b) activating said crosslinking moiety, whereby a covalent crosslink occurs between said first and second crosslinkable probes and said target nucleic acid; and
c) detecting the presence of a crosslinked nucleic acid pair as indicative of the presence of said target sequence in said sample. - View Dependent Claims (20, 21, 22, 23, 24)
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Specification