Detection of nucleic acids by target-specific hybrid capture method
First Claim
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1. A method of detecting at least one target nucleic acid comprising:
- a) forming a plurality of enriched target nucleic acids comprisingi) mixing the at least one target nucleic acid with a nucleic acid probe complementary to at least a portion of the at least one target nucleic acid to form a target DNA;
RNA hybrid, wherein when the target nucleic acid is RNA, the probe is DNA, and wherein when the target nucleic acid is DNA, the probe is RNA;
ii) capturing the target DNA;
RNA hybrid onto a solid support; and
iii) removing any nucleic acid that does not form the target DNA;
RNA hybrid or that is not captured onto the solid support to form the plurality of enriched target nucleic acids;
b) amplifying the plurality of enriched target nucleic acids from step a) to form a plurality of amplified targets; and
c) detecting the at least one target nucleic acid in the plurality of amplified targets byi) mixing the plurality of amplified targets with a selectable oligonucleotide and a nucleic acid probe, wherein the selectable oligonucleotide hybridizes to a first portion of the amplified targets and wherein the nucleic acid probe is complementary to a second portion of the amplified targets wherein if the amplified target is DNA, the selectable oligonucleotide and the nucleic acid probe is RNA and if the amplified target is RNA, the selectable oligonucleotide and the nucleic acid probe is DNA to form a DNA;
RNA hybrid target/oligonucleotide/probe complex, wherein the first portion of the amplified targets and the second portion of the amplified targets are different; and
ii) detecting the DNA;
RNA hybrid target/oligonucleotide/probe complex by a DNA;
RNA hybrid-specific binding agent, to detect at least one 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.
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Citations
26 Claims
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1. A method of detecting at least one target nucleic acid comprising:
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a) forming a plurality of enriched target nucleic acids comprising i) mixing the at least one target nucleic acid with a nucleic acid probe complementary to at least a portion of the at least one target nucleic acid to form a target DNA;
RNA hybrid, wherein when the target nucleic acid is RNA, the probe is DNA, and wherein when the target nucleic acid is DNA, the probe is RNA;ii) capturing the target DNA;
RNA hybrid onto a solid support; andiii) removing any nucleic acid that does not form the target DNA;
RNA hybrid or that is not captured onto the solid support to form the plurality of enriched target nucleic acids;b) amplifying the plurality of enriched target nucleic acids from step a) to form a plurality of amplified targets; and c) detecting the at least one target nucleic acid in the plurality of amplified targets by i) mixing the plurality of amplified targets with a selectable oligonucleotide and a nucleic acid probe, wherein the selectable oligonucleotide hybridizes to a first portion of the amplified targets and wherein the nucleic acid probe is complementary to a second portion of the amplified targets wherein if the amplified target is DNA, the selectable oligonucleotide and the nucleic acid probe is RNA and if the amplified target is RNA, the selectable oligonucleotide and the nucleic acid probe is DNA to form a DNA;
RNA hybrid target/oligonucleotide/probe complex, wherein the first portion of the amplified targets and the second portion of the amplified targets are different; andii) detecting the DNA;
RNA hybrid target/oligonucleotide/probe complex by a DNA;
RNA hybrid-specific binding agent, to detect at least one target nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 22, 25, 26)
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18. A method of detecting each of at least one target nucleic acid comprising:
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a) hybridizing the at least one target nucleic acid to a nucleic acid probe that is complementary to at least a portion of the target nucleic acid, to form a target DNA;
RNA hybrid wherein when the target nucleic acid is RNA, the probe is DNA, and wherein the target nucleic acid is DNA, the probe is RNA;b) capturing the target DNA;
RNA hybrid with a DNA;
RNA hybrid-specific antibody conjugated to a solid support;c) separating unbound target nucleic acid from the captured target DNA;
RNA hybrid;d) amplifying the captured target DNA;
RNA hybrid to form a plurality of amplified targets, wherein the amplifying uses random primers and DNA polymerase;e) hybridizing a nucleic acid probe complementary to a first portion of the amplified targets to form an amplified DNA;
RNA hybrid;I) hybridizing an oligonucleotide conjugated to a solid support to a second portion of the amplified DNA;
RNA hybrid, wherein the solid support is selectable; andg) selecting amplified DNA;
RNA hybrid by the selectable solid support; andh) detecting the at least one target nucleic acid by binding a DNA;
RNA hybrid-specific binding agent to the amplified DNA;
RNA hybrid, wherein the presence of the amplified DNA;
RNA hybrid is indicative of the presence of the target nucleic acid. - View Dependent Claims (19, 20)
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21. A multiplex method of detecting at least one target DNA comprising:
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a) hybridizing at least one target DNA to at least one first RNA probe which is complementary to at least a portion of the at least one target DNA, to form at least one DNA;
RNA hybrid;b) capturing the at least one DNA;
RNA hybrid with at least one DNA;
RNA hybrid-specific antibody conjugated to a bead;c) separating unbound nucleic acid from the at least one DNA;
RNA hybrid, to form at least one enriched target;d) amplifying the at least one enriched target DNA, to form at least one amplified target DNA, using random primers and DNA polymerase; e) hybridizing a second RNA probe complementary to a first portion of the amplified target DNA to form an amplified DNA;
RNA hybrid;f) hybridizing a DNA oligonucleotide to a second portion of the amplified target DNA, wherein the DNA oligonucleotide is conjugated to at least one selectable bead; and g) detecting the at least one amplified target DNA by binding a DNA;
RNA hybrid-specific antibody to the amplified DNA;
RNA hybrid and selecting the amplified target based on the particular selectable bead.
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23. A method of detecting at least one target nucleic acid comprising:
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a) forming a plurality of enriched target nucleic acids comprising i) mixing the at least one target nucleic acid with a nucleic acid probe complementary to at least one unique region of the at least one target nucleic acid to form a target DNA;
RNA hybrid whereby the probe comprises a nucleic acid sequence that hybridizes to said at least one unique region, and wherein when the target nucleic acid is RNA, the probe is DNA, and wherein when the target nucleic acid is DNA, the probe is RNA;ii) capturing the target DNA;
RNA hybrid onto a solid support; andiii) removing any nucleic acid that does not form the target DNA;
RNA hybrid or that is not captured onto the solid support to form the plurality of enriched target nucleic acids;b) amplifying the plurality of enriched target nucleic acids from step a) to form a plurality of amplified targets; and c) detecting the at least one target nucleic acid in the plurality of amplified targets by i) mixing the plurality of amplified targets with a selectable oligonucleotide and a nucleic acid probe, wherein the selectable oligonucleotide hybridizes to a first portion of the amplified targets and wherein the nucleic acid probe is complementary to a second portion of the amplified targets wherein if the amplified target is DNA, the selectable oligonucleotide and the nucleic acid probe is RNA and if the amplified target is RNA, the selectable oligonucleotide and the nucleic acid probe is DNA to form a DNA;
RNA hybrid target/oligonucleotide/probe complex;
wherein the first portion of the amplified targets and the second portion of the amplified targets are different; andii) detecting the DNA;
RNA hybrid target/oligonucleotide/probe complex by a DNA;
RNA hybrid-specific binding agent, to detect at least one target nucleic acid.
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24. A method of detecting at least one target nucleic acid comprising:
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a) forming a plurality of enriched target nucleic acids comprising i) mixing the at least one target nucleic acid with a nucleic acid probe complementary to at least a portion of the at least one target nucleic acid to form a target DNA;
RNA hybrid, wherein when the target nucleic acid is RNA, the probe is DNA, and wherein when the target nucleic acid is DNA, the probe is RNA; and
wherein the probe is specific to the target nucleic acid;ii) capturing the target DNA;
RNA hybrid onto a solid support; andiii) removing any nucleic acid that does not form the target DNA;
RNA hybrid or that is not captured onto the solid support to form the plurality of enriched target nucleic acids;b) amplifying the plurality of enriched target nucleic acids from step a) to form a plurality of amplified targets; and c) detecting the at least one target nucleic acid in the plurality of amplified targets by i) mixing the plurality of amplified targets with a selectable oligonucleotide and a nucleic acid probe, wherein the selectable oligonucleotide hybridizes to a first portion of the amplified targets and wherein the nucleic acid probe is complementary to a second portion of the amplified targets wherein if the amplified target is DNA, the selectable oligonucleotide and the nucleic acid probe is RNA and if the amplified target is RNA, the selectable oligonucleotide and the nucleic acid probe is DNA to form a DNA;
RNA hybrid target/oligonucleotide/probe complex;
wherein the first portion of the amplified targets and the second portion of the amplified targets are different; andii) detecting the DNA;
RNA hybrid target/oligonucleotide/probe complex by a DNA;
RNA hybrid-specific binding agent, to detect at least one target nucleic acid.
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Specification