Methods and compositions to detect nucleic acids in a biological sample
First Claim
1. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:
- providing a sample containing a target nucleic acid, mixing the sample with a nucleic acid capture probe that forms under hybridizing conditions a partially double-stranded hairpin structure made up of an internal target-complementary sequence, flanked by a capture region, and a terminal region that binds to the capture region to form a double-stranded stem portion of the hairpin structure in which the target-complementary region forms a substantially single-stranded loop portion, specifically hybridizing the target-complementary sequence of the capture probe to a target sequence in the target nucleic acid, binding the capture region to an immobilized probe attached to a capture support by binding together members of a specific binding pair, thereby forming a capture hybrid made up of the target nucleic acid, the capture probe, and the immobilized probe attached to the capture support, separating the capture hybrid attached to the capture support from sample components, releasing the target nucleic acid from the capture hybrid, then specifically hybridizing a detection probe to the target nucleic to form a detection hybrid, and detecting a signal produced from the detection hybrid to indicate the presence of the target nucleic acid in the sample.
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Accused Products
Abstract
Methods of the invention separate a target nucleic acid from a sample by using at least one capture probe oligonucleotide that contains a target-complementary region and a member of a specific binding pair that attaches the target nucleic acid to an immobilized probe on a capture support, thus forming a capture hybrid that is separated from other sample components before the target nucleic acid is released from the capture support and hybridized to a detection probe to form a detection hybrid that produces a detectable signal that indicates the presence of the target nucleic acid in the sample. Compositions for practicing the methods of the invention include a capture probe oligonucleotide made up a target-complementary region sequence and a covalently linked capture region sequence that includes a member of a specific binding pair.
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Citations
20 Claims
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1. A method of detecting the presence of a target nucleic acid in a sample comprising the steps of:
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providing a sample containing a target nucleic acid, mixing the sample with a nucleic acid capture probe that forms under hybridizing conditions a partially double-stranded hairpin structure made up of an internal target-complementary sequence, flanked by a capture region, and a terminal region that binds to the capture region to form a double-stranded stem portion of the hairpin structure in which the target-complementary region forms a substantially single-stranded loop portion, specifically hybridizing the target-complementary sequence of the capture probe to a target sequence in the target nucleic acid, binding the capture region to an immobilized probe attached to a capture support by binding together members of a specific binding pair, thereby forming a capture hybrid made up of the target nucleic acid, the capture probe, and the immobilized probe attached to the capture support, separating the capture hybrid attached to the capture support from sample components, releasing the target nucleic acid from the capture hybrid, then specifically hybridizing a detection probe to the target nucleic to form a detection hybrid, and detecting a signal produced from the detection hybrid to indicate the presence of the target nucleic acid in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of detecting the presence of a target nucleic acid present in a sample comprising the steps of:
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providing a sample containing a target nucleic acid, mixing the sample with a capture probe that is at least a partially double-stranded structure made up of a first strand and a second strand of nucleic acid, wherein the first strand includes a target-complementary region and a capture region, and the second strand contains a sequence complementary to a sequence of the first strand, specifically hybridizing the target-complementary region of the capture probe to a target sequence in the target nucleic acid, binding the capture region to an immobilized probe attached to a capture support, thereby forming a capture hybrid made up of the target nucleic acid, the first strand of the capture probe, and the immobilized probe attached to the capture support, separating the capture hybrid attached to the capture support from other sample components, releasing the target nucleic acid from the capture hybrid, then specifically hybridizing a detection probe to the target nucleic acid to form a detection hybrid, and detecting a signal produced from the detection hybrid, thereby indicating the presence of the target nucleic acid in the sample. - View Dependent Claims (11, 12)
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- 13. A nucleic acid capture probe that forms at least a partially double-stranded structure under hybridizing conditions, comprising a target-complementary sequence and a capture region that binds to an immobilized probe by using members of a specific binding pair.
Specification