Methods and compositions to detect nucleic acids in a biological sample
View Patent ↗Kits, reaction mixtures and methods for separating a target nucleic acid from a sample by using at least one hairpin capture probe oligonucleotide that has the structure 5′-X.sub.n a′ b′ c′ Y.sub.n-3′, wherein X and Y each comprise nucleic acid sequences that can form a double stranded stem portion, one of X or Y is a capture sequence that is a first member of a specific binding pair and the other of X or Y is a terminal sequence of the hairpin capture probe, and a′ b′ c′ comprises a target-complementary sequence flanked by X and Y to thereby form a loop portion of the hairpin, 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 that hybridizes specifically to the same sequence that is at least partially hybridized by the a′ b′ c′ portion of the capture probe, thus forming a detectable detection hybrid to indicate the presence of the target nucleic acid in the sample.
1. A kit for the detection of a target nucleic acid, wherein the kit comprises: a capture probe having the structure 5′-X.sub.n a′ b′ c′ Y.sub.n-3′, wherein X and Y each comprise nucleic acid sequences that can form a double stranded portion of a hairpin structure and wherein one of X or Y is a capture sequence further comprising a first member of a specific binding pair and the other of X or Y is a terminal sequence of the hairpin capture probe, and wherein a′ b′ c′ comprises a target-complementary sequence that hybridizes specifically to a target sequence in the target nucleic acid and is flanked by X and Y to thereby form a loop portion of the hairpin structure; and a detection probe that hybridizes specifically to a target sequence that overlaps or is the same as the target sequence hybridized by the target-complementary sequence of the capture probe.
2. The kit of claim 1 , wherein the detection probe hybridizes specifically to the same target sequence hybridized by the target-complementary sequence of the capture probe.
3. The kit of claim 1 , wherein the detection probe comprises a target-complementary sequence that is a′ b′ c′.
4. The kit of claim 1 that further includes an immobilized probe attached to a capture support, wherein the immobilized probe includes a second member of a specific binding pair that binds specifically to the capture probe's first member of a specific binding pair.
5. The kit of claim 4 , wherein the first and second specific binding pair members are substantially complementary sequences that hybridize the capture probe to the immobilized probe under hybridizing conditions.
6. The kit of claim 5 , wherein the first member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.
7. The kit of claim 5 , wherein the second member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.
8. The kit of claim 1 , wherein said target complementary sequence is synthesized with 2′-O-methyl linkages.
9. The kit of claim 1 , wherein Y is the capture sequence that forms the first member of the specific binding pair.
10. The kit of claim 9 , wherein the capture probe has the structure 5′-X.sub.n a′b′c′ poly(A).sub.10-30.
11. A reaction mixture for the detection of a target nucleic acid, wherein the reaction mixture comprises: a capture probe having the structure 5′-X.sub.n a′ b′ c′ Y.sub.n-3′, wherein X and Y each comprise nucleic acid sequences that can form a double stranded portion of a hairpin structure and wherein one of X or Y is a capture sequence further comprising a first member of a specific binding pair and the other of X or Y is a terminal sequence of the hairpin capture probe, and wherein a′ b′ c′ comprises a target-complementary sequence that hybridizes specifically to a target sequence in the target nucleic acid and is flanked by X and Y to thereby form a loop portion of the hairpin structure; and a detection probe that hybridizes specifically to a target sequence that overlaps or is the same as the target sequence hybridized by the target-complementary sequence of the capture probe.
12. The reaction mixture of claim 11 , wherein the detection probe hybridizes specifically to the same target sequence hybridized by the target-complementary sequence of the capture probe.
13. The reaction mixture of claim 11 , wherein the detection probe comprises a target-complementary sequence that is a′ b′ c′.
14. The reaction mixture of claim 11 , wherein the first member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.
15. The reaction mixture of claim 11 , wherein the second member of the specific binding pair is a sequence from 10 to 30 nucleobases in length and is substantially a poly-A sequence.
16. The reaction mixture of claim 11 , wherein said target complementary sequence is synthesized with 2′-O-methyl linkages.
17. The reaction mixture of claim 11 , wherein Y is the capture sequence that forms the first member of the specific binding pair.
18. The reaction mixture of claim 17 , wherein the capture probe has the structure 5′-X.sub.n a′b′c′ poly(A).sub.10-30.
19. The reaction mixture of claim 11 , wherein the detection probe is labeled with a chemiluminescent compound.
20. The reaction mixture of claim 19 , wherein the chemiluminescent compound is an acridinium ester.
21. The reaction mixture of claim 11 , wherein the detection probe is labeled with a fluorescent compound.
22. The reaction mixture of claim 21 , wherein the detection probe is a molecular torch or a molecular beacon.
23. The reaction mixture of claim 11 , wherein the reaction mixture contains the target nucleic acid, and wherein the detection probe is hybridized to the target nucleic acid.