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US 2002/0172946 A1Published application
Nucleic acid detection methods using universal priming
AssigneesThe Regents of the University of California, Illumina, Inc.
InventorsJian-Bing Fan, Xiang-Dong Fu
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Abstract
The present invention is directed to providing sensitive and accurate assays for gene detection, genome-wide gene expression profiling and alternative splice monitoring with a minimum or absence of target-specific amplification.
Claims19
A method of detecting a first target sequence comprising a poly(A) sequence in a sample comprising:
- a) hybridizing a first probe to said target sequence to form a first hybridization complex, said first probe comprising:
- i) an upstream universal priming site (UUP);
- ii) an adapter sequence;
- iii) a first target-specific sequence;
- and
- iv) a downstream universal priming site (DUP);
- wherein said poly(A) sequence remains single-stranded;
- b) contacting said first hybridization complex with a support comprising a poly(T) sequence, such that said poly(A) sequence hybridizes with said poly(T) sequence;
- c) removing unhybridized first probe sequences;
- d) denaturing said first hybridization complex;
- e) amplifying said first probe to generate a plurality of amplicons;
- f) contacting said amplicons with an array of capture probes to form assay complexes;
- and
- g) detecting said assay complexes.
11 more elements
A method of detecting a first target sequence comprising a first target domain, a second adjacent target domain and a poly(A) sequence, said method comprising:
- a) hybridizing a first probe comprising:
- i) an upstream universal priming site (UUP);
- and
- ii) a first target-specific sequence substantially complementary to said first target domain;
- to said first target domain;
- b) hybridizing a second probe comprising:
- iii) a second target-specific sequence substantially complementary to said second target domain;
- iv) a downstream universal priming site (DUP);
- wherein at least one of said first and second probes comprises at least a first adapter sequence, said poly(A) sequence remains single-stranded, and said target sequence and said first and second probes form a ligation complex;
- c) contacting said ligation complex with a ligase to form a ligated complex;
- d) contacting said ligated complex with a support comprising a poly(T) sequence, such that said poly(A) sequence hybridizes with said poly(T) sequence;
- e) removing unhybridized first and second probe sequences;
- f) denaturing said ligation complex;
- g) amplifying the ligated first and second probes to generate a plurality of amplicons;
- h) contacting said amplicons with an array of capture probes to form assay complexes;
- and
- i) detecting said assay complexes.
14 more elements
Dependencies are inferred from the claim text.