Universal tag assay
First Claim
1. A method for detecting a target nucleotide sequence in a sample, comprising:
- a) generating at least one tagged molecule comprising at least one identifier tag selected as an identifier for said target nucleotide sequence, wherein said identifier tag is generated only when said target nucleotide sequence corresponding to said identifier tag is present in said sample;
b) incubating said at least one tagged molecule with a universal detector having at least one detection probe complementary to said identifier tag; and
c) measuring hybridization of any said identifier tag to any said detection probe complementary to said identifier tag;
wherein said hybridization of any said identifier tag to any said detection probe complementary to said identifier tag indicates said target nucleotide sequence corresponding to said identifier tag is present in said sample.
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Abstract
The present invention disclosure provides methods and compositions for a universal tag assay wherein a universal detector having detection probes is incubated with tagged molecules having identifier tags corresponding to targets, and hybridization of an identifier tag to a complementary detection probe indicates the presence of the corresponding target in the material being assayed. In particular, the invention disclosure provides methods and compositions for detecting target nucleotide sequences in a sample by target-dependent manipulations that generate tagged molecules having identifier tags corresponding to target nucleotide sequence, where incubation of tagged molecules with a universal detector having detection probes permits hybridization of identifier tags to complementary detection probes, thereby indicating the presence of the target nucleotide sequence corresponding to each identifier tag. Preferred embodiments include use of the universal tag assay for detecting variant sequences including single nucleotide polymorphisms (SNPs), allelic variants, and splice variants. Preferred embodiments further include the use of ruthenium amperometry to detect hybridization of tagged DNA or RNA molecules to detection probes immobilized on a universal detector, preferably a universal chip having gold or carbon electrodes.
202 Citations
51 Claims
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1. A method for detecting a target nucleotide sequence in a sample, comprising:
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a) generating at least one tagged molecule comprising at least one identifier tag selected as an identifier for said target nucleotide sequence, wherein said identifier tag is generated only when said target nucleotide sequence corresponding to said identifier tag is present in said sample;
b) incubating said at least one tagged molecule with a universal detector having at least one detection probe complementary to said identifier tag; and
c) measuring hybridization of any said identifier tag to any said detection probe complementary to said identifier tag;
wherein said hybridization of any said identifier tag to any said detection probe complementary to said identifier tag indicates said target nucleotide sequence corresponding to said identifier tag is present in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for detecting a target nucleotide sequence in a sample, comprising:
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a) obtaining template comprising said target nucleotide sequence or complement thereof;
b) amplifying said template to generate at least one tagged molecule comprising at least one identifier tag selected as an identifier for said target nucleotide sequence;
c) incubating said at least one tagged molecule with a universal detector comprising detection probes coupled to a detection means, said detection probes comprising at least one detection probe complementary to said identifier tag; and
d) detecting hybridization of any said identifier tag to any said detection probe complementary to said identifier tag;
wherein hybridization of any said identifier tag to any said complementary detection probe indicates the presence of the corresponding target nucleotide sequence in the sample being assayed. - View Dependent Claims (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 for detecting a target nucleotide sequence in a sample, comprising:
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a) obtaining template comprising said target nucleotide sequence or complement thereof;
b) providing at least one pair of ligation primers comprising a first ligation primer having a 3′
portion of sequence complementary to a portion of said template and a second ligation primer having a 5′
portion of sequence complementary to a contiguous portion of said template, wherein each ligation primer of said pair of ligation primers further comprises exogenous sequence not complementary to said template;
c) incubating said at least one pair of ligation primers with said template under conditions such that said first and second ligation primers will hybridize to said template, said 3′
end of said first ligation primer hybridizing adjacently to said 5′
end of said second ligation primer;
d) ligating said 3′
end of said first ligation primer to said 5′
end of said second ligation primer, thereby generating a ligation product comprising a copy or complement of said target nucleotide sequence and further comprising exogenous 3′ and
5′
sequence not complementary to said template;
e) providing a rolling circle (RC) probe comprising sequence complementary to said ligation product and further comprising sequence complementary to an identifier tag selected to serve as an identifier for said target nucleotide sequence, wherein said sequence complementary to said ligation product comprises sequence complementary to said target nucleotide sequence or complement thereof, flanked by sequence complementary to said exogenous sequence of said ligation product;
f) incubating said RC probe with said ligation product under conditions such that said RC probe will hybridize to said ligation product;
g) providing polymerase enzyme under conditions such that said polymerase replicates said RC probe, generating at least one amplification product comprising a tagged molecule, wherein said tagged molecule comprises repeating copies of said identifier tag and repeating copies of said target nucleotide sequence or complement thereof;
h) incubating each said amplification product with a universal detector comprising detection probes coupled to a detection means, said detection probes comprising at least one detection probe complementary to said identifier tag; and
i) detecting hybridization of any said identifier tag in said amplification product to any said detection probe complementary to said identifier tag;
wherein hybridization of any said identifier tag to any said complementary detection probe indicates the presence of the corresponding target nucleotide sequence in the sample being assayed. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47)
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48. A universal tag assay comprising:
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a) a set of minimally cross-hybridizing tags and probes selected such that at least one tag will serve as an identifier tag for a target in a sample being assayed and each said identifier tag has at least one complementary detection probe in said set;
b) at least one tagged molecule comprising at least one said identifier tag, wherein said identifier tag is generated only when said target corresponding to said identifier tag is present in said sample being assayed; and
c) a universal detector comprising at least one said detection probe coupled to a detection means such that hybridization of said any identifier tag to any said complementary detection probe can be measured;
wherein measuring hybridization of any said identifier tag to any said complementary detection probe indicates that said target corresponding to said identifier tag is present in said sample being assayed. - View Dependent Claims (49, 50, 51)
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Specification