Nucleic acid detection methods using universal priming
First Claim
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1. A method of detecting a splice junction sequence in a plurality of target pre-mRNA sequences, said method comprising:
- a) hybridizing said plurality of target pre-mRNA sequences to a plurality of first splice junction specific probes and forming first hybrids wherein each of said plurality of target pre-mRNA sequences comprises a first target domain, a second target domain and a poly(A) sequence, said first target domain and said second target domain are adjacent each other and said first target domain comprises a splice junction sequence, and each of said plurality of first splice junction specific probes comprises;
i) an upstream universal priming site (UUP), wherein each of said first splice junction specific probes has the same UUP;
ii) at least one adapter; and
iii) a first target-specific sequence substantially complementary to said first target domain, and said upstream universal priming site (UUP) locates in the upstream of said adapter and said first target-specific sequence;
b) hybridizing said plurality of target pre-mRNA sequences of said first hybrids to a plurality of second probes and forming second hybrids wherein each of said second hybrids comprises a said target pre-mRNA sequence, a said first splice junction specific probe and a said second probe and each of said plurality of second probes comprises;
iv) a second target-specific sequence substantially complementary to said second target domain;
v) a downstream universal priming site (DUP), wherein each of said second probes has the same DUP, and said downstream universal priming site (DUP) locates in the downstream of said second target-specific sequence, and wherein, in each of said plurality of first and second hybrids, said poly(A) sequence remains single-stranded;
c) contacting said second hybrids with a ligase and forming ligation complexes comprising ligated probes, each of said ligated probes comprising a said first splice junction specific probe and a said second probe;
d) contacting said ligation complexes with a support comprising a poly(T) sequence, such that said poly(A) sequence hybridizes with said poly(T) sequence;
e) removing unhybridized said first splice junction specific probes and unhybridized said second probes;
f) denaturing said ligation complexes;
g) amplifying the ligated probes using universal primers that hybridize to the UUP and DUP, or complement thereof, and generating a plurality of amplicons each comprising a said adapter;
h) contacting said amplicons with an array of capture probes to form assay complexes, wherein said capture probes bind to said adapter; and
i) detecting said assay complexes wherein the detection of said assay complexes is an indication of the presence of said splice junction sequence in the pre-mRNA sequences.
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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.
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Citations
14 Claims
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1. A method of detecting a splice junction sequence in a plurality of target pre-mRNA sequences, said method comprising:
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a) hybridizing said plurality of target pre-mRNA sequences to a plurality of first splice junction specific probes and forming first hybrids wherein each of said plurality of target pre-mRNA sequences comprises a first target domain, a second target domain and a poly(A) sequence, said first target domain and said second target domain are adjacent each other and said first target domain comprises a splice junction sequence, and each of said plurality of first splice junction specific probes comprises; i) an upstream universal priming site (UUP), wherein each of said first splice junction specific probes has the same UUP; ii) at least one adapter; and iii) a first target-specific sequence substantially complementary to said first target domain, and said upstream universal priming site (UUP) locates in the upstream of said adapter and said first target-specific sequence; b) hybridizing said plurality of target pre-mRNA sequences of said first hybrids to a plurality of second probes and forming second hybrids wherein each of said second hybrids comprises a said target pre-mRNA sequence, a said first splice junction specific probe and a said second probe and each of said plurality of second probes comprises; iv) a second target-specific sequence substantially complementary to said second target domain; v) a downstream universal priming site (DUP), wherein each of said second probes has the same DUP, and said downstream universal priming site (DUP) locates in the downstream of said second target-specific sequence, and wherein, in each of said plurality of first and second hybrids, said poly(A) sequence remains single-stranded; c) contacting said second hybrids with a ligase and forming ligation complexes comprising ligated probes, each of said ligated probes comprising a said first splice junction specific probe and a said second probe; d) contacting said ligation complexes with a support comprising a poly(T) sequence, such that said poly(A) sequence hybridizes with said poly(T) sequence; e) removing unhybridized said first splice junction specific probes and unhybridized said second probes; f) denaturing said ligation complexes; g) amplifying the ligated probes using universal primers that hybridize to the UUP and DUP, or complement thereof, and generating a plurality of amplicons each comprising a said adapter; h) contacting said amplicons with an array of capture probes to form assay complexes, wherein said capture probes bind to said adapter; and i) detecting said assay complexes wherein the detection of said assay complexes is an indication of the presence of said splice junction sequence in the pre-mRNA sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of detecting a splice junction sequence in a target pre-mRNA sequence, said method comprising:
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a) hybridizing said target pre-mRNA sequence to a first splice junction specific probe and forming a first hybrid wherein said target pre-mRNA sequence comprises a first target domain, a second target domain and a poly(A) sequence, said first target domain and said second target domain are adjacent each other and said first target domain comprises a splice junction sequence and said first splice junction specific probe comprises; i) an upstream universal priming site (UUP); ii) at least one adapter; and iii) a first target-specific sequence substantially complementary to said first target domain, wherein said upstream universal priming site (UUP) locates in the upstream of said adapter and said first target-specific sequence, and said adapter is exogenous to said first target-specific sequence; b) hybridizing said target pre-mRNA sequence of said first hybrid to a sequence of a second probe and forming a second hybrid wherein said second hybrid comprises a said target pre-mRNA sequence, a said first splice junction specific probe and a said second probe and said second probe comprises; iv) a second target-specific sequence substantially complementary to said second target domain; v) a downstream universal priming site (DUP), wherein said downstream universal priming site (DUP) locates in the downstream of said second target-specific sequence, and said poly(A) sequence remains single-stranded in each of said first and second hybrids; c) contacting said second hybrid with a ligase and forming a ligation complex comprising ligated probes, each of said ligated probes comprising a said first splice junction specific probe and a said second probe; d) contacting said ligation complex with a support comprising a poly(T) sequence, such that said poly(A) sequence hybridizes with said poly(T) sequence; e) removing unhybridized said first splice junction specific probe and unhybridized said second probe; f) denaturing said ligation complex; g) amplifying the ligated probes using universal primers that hybridize to the UUP and DUP, or complement thereof, and generating a plurality of amplicons each comprising a said adapter; h) contacting said amplicons with an array of capture probes to form assay complexes, wherein said capture probes bind to said adapter; and i) detecting said assay complexes wherein the detection of said assay complexes is an indication of the presence of said splice junction sequence in the pre-mRNA sequence.
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14. A method of detecting a splice junction sequence in a plurality of target pre-mRNA sequences, said method comprising:
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a) hybridizing said plurality of target pre-mRNA sequences to aplurality of first splice junction specific probes and form first hybrids wherein each of said plurality of target pre-mRNA sequences comprises a first target domain, a second target domain and a poly(A) sequence, said first target domain and said second target domain are adjacent each other and said first target domain comprises a splice junction sequence; and
each of said plurality of first splice junction specific probes comprises;i) an upstream universal priming site (UUP), wherein each of said first splice junction specific probes has the same UUP; ii) at least one adapter; and iii) a first target-specific sequence substantially complementary to said first target domain, and said upstream universal priming site (UUP) locates in the upstream of said adapter and said first target-specific sequence; b) hybridizing said plurality of target pre-mRNA sequences of said first hybrids to a plurality of second probes and forming second hybrids wherein each of said second hybrids comprises a said target pre-mRNA sequence, a said first splice junction specific probe and a said second probe and each of said plurality of second probes comprises; iv) a second target-specific sequence substantially complementary to said second target domain; v) a downstream universal priming site (DUP), wherein each of said second probes has the same DUP, and said downstream universal priming site (DUP) locates in the downstream of said second target-specific sequence; c) hybridizing said second hybrids to a poly(T) sequence on a solid support and forming third hybrids, such that said poly(A) sequence hybridizes with said poly(T) sequence; d) contacting said third hybrids with a ligase and forming ligation complexes comprising ligated probes, each of said ligated probes comprising a said first splice junction specific probe and a said second probe; e) removing unhybridized said first splice junction specific probes and unhybridized said second probes; f) denaturing said ligation complexes; g) amplifying the ligated probes using universal primers that hybridize to the UUP and DUP, or complement thereof, and generating a plurality of amplicons each comprising a said adapter; h) contacting said amplicons with an array of capture probes to form assay complexes, wherein said capture probes bind to said adapter; and i) detecting said assay complexes wherein the detection of said assay complexes is an indication of the presence of said splice junction sequence in the pre-mRNA sequences.
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Specification