Detection of nucleic acids by type-specific hybrid capture method
First Claim
Patent Images
1. A method of detecting a target nucleic acid consisting essentially of:
- a) hybridizing a single-stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other, wherein said hybridization forms an RNA/DNA hybrid between said signal sequence probe and the target nucleic acid; and
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 RNA/DNA hybrid by binding an antibody that recognizes the RNA/DNA hybrid to said hybrid, wherein said antibody is detectably labeled.
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Abstract
Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but also highly specific and capable of discriminating highly homologous nucleic acid sequences. The method produces DNA/RNA hybrids which can be detected by a variety of methods.
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Citations
60 Claims
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1. A method of detecting a target nucleic acid consisting essentially of:
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a) hybridizing a single-stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other, wherein said hybridization forms an RNA/DNA hybrid between said signal sequence probe and the target nucleic acid; and 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 RNA/DNA hybrid by binding an antibody that recognizes the RNA/DNA hybrid to said hybrid, wherein said antibody is detectably labeled. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 27, 54, 57, 58)
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18. A method of detecting a target nucleic acid consisting essentially of:
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a) hybridizing a single stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other, wherein the signal sequence probe comprises a DNA/RNA hybrid region, wherein said hybridization forms a complex; and b) adding a blocker probe after the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes; and c) detecting said complex. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 55, 59)
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28. A method of detecting a target nucleic acid comprising:
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a) hybridizing a single-stranded or partially single-stranded target nucleic acid to a capture sequence probe and a signal sequence probe to form double-stranded hybrids between said probes and the target nucleic acid; and b) adding a blocker probe to the hybridization reaction, wherein said blocker probe hybridizes to excess non-hybridized capture sequence probes; and c) binding the hybrid to a solid phase forming a bound hybrid; and d) detecting the bound hybrid, wherein the capture sequence probe and the signal sequence probe hybridize to non-overlapping regions within the target nucleic acid and not to each other, wherein step c) is carried out at about 20°
C. to about 90°
C.- View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 60)
a sequence capable of hybridizing to the 3′
side of a target sequence to which a capture sequence probe is capable of hybridizing.
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Specification