Detection of nucleic acids by type-specific hybrid capture method
First Claim
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1. A method of detecting a target nucleic acid comprising:
- a) hybridizing a single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe to form a hybrid complex comprising double-stranded hybrids between said capture sequence probe and a portion of the target nucleic acid, and between said signal sequence probe and a portion of the target nucleic acid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and do not hybridize to each other, wherein the signal sequence probe is unlabeled; and
b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes;
c) capturing the capture sequence probe;
target portion of said hybrid complex to form a bound hybrid complex; and
d) detecting the bound double-stranded hybrid complex, thereby detecting the target nucleic acid.
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Abstract
Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid target sequences. The method produces DNA-RNA hybrids which can be detected by a variety of methods.
77 Citations
34 Claims
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1. A method of detecting a target nucleic acid comprising:
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a) hybridizing a single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe to form a hybrid complex comprising double-stranded hybrids between said capture sequence probe and a portion of the target nucleic acid, and between said signal sequence probe and a portion of the target nucleic acid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and do not hybridize to each other, wherein the signal sequence probe is unlabeled; and b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes; c) capturing the capture sequence probe;
target portion of said hybrid complex to form a bound hybrid complex; andd) detecting the bound double-stranded hybrid complex, thereby detecting the target nucleic acid. - View Dependent Claims (3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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2. A method of detecting a target nucleic acid comprising:
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a) hybridizing a single-stranded target nucleic acid to an immobilized capture sequence probe and a signal sequence probe to form a hybrid complex comprising double-stranded hybrids between said immobilized capture sequence probe and a portion of the target nucleic acid, and between said signal sequence probe and a portion of the target nucleic acid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and do not hybridize to each other; b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes; and c) detecting the immobilized double-stranded hybrid complex, thereby detecting the target nucleic acid. - View Dependent Claims (4)
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29. A method of detecting a target nucleic acid comprising:
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a) hybridizing a single-stranded target nucleic acid to a capture sequence probe, a bridge probe and a signal sequence probe to form a hybrid complex comprising double-stranded hybrids between said capture sequence probe and a portion of the target nucleic acid, and between said bridge probe and a portion of the target nucleic acid, wherein the capture sequence probe and the bridge probe each hybridize to non-overlapping regions within the target nucleic acid and do not hybridize to each other, and the signal sequence probe hybridizes to the bridge probe and does not hybridize to the target nucleic acid and the capture sequence probe; b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes; c) capturing the hybrid complex to form a bound double-stranded hybrid complex; and d) detecting the bound double-stranded hybrid complex, thereby detecting the target nucleic acid. - View Dependent Claims (30, 31, 32, 33, 34)
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Specification