Methods and compositions for detecting target nucleic acids
The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more ligation probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligation product that is directly detected or that is amplified to produce amplicons that are then detected. The present invention also provides methods to stabilize sample RNA so that degradation does not significantly affect the results of the analysis.
1. A method of detecting a plurality of different target nucleic acids in a target sample, wherein each target nucleic acid comprises an adjacent first and a second target domain, said method comprising:
a) providing a reaction mixture comprising said target sample in lysis buffer comprising 2 to 6 molar guanidinium salt;
b) contacting said reaction mixture with a plurality of different probes sets, each probe set comprising:
i) a first nucleic acid ligation probe comprising:
1) a first probe domain complementary to a first target domain of said one a target nucleic acid;
2) a first primer sequence; and
3) a 5′-ligation moiety; and
ii) a second nucleic acid ligation probe comprising:
1) a second probe domain complementary to a second target domain of said one target nucleic acid;
2) a second primer sequence; and
3) a 3′ ligation moiety;
wherein one of said first or second nucleic acid ligation probes comprises a detectable label and the other comprises one of a binding partner pair;
c) ligating said first and second ligation probes in the absence of a ligase enzyme to form a plurality of different ligation products;
d) contacting said ligation products with a solid support comprising the other of said binding partner pair such that said ligation products are captured on said solid support;
e) washing said solid support;
f) amplifying said different ligation products; and
g) detecting the presence of said ligation products.
2. A method according to claim 1 wherein said detectable label comprises a variable spacer sequence and said plurality of different probe sets have different length variable spacer sequences.
3. A method according to claim 2 , wherein said detecting is done using said variable spacer sequence.
4. A method according to claim 1 wherein said target nucleic acids are RNA.
5. A method according to claim 1 wherein one of said first and second ligation probes further comprises one of a binding partner pair, and prior to said amplifying, a bead comprising the other of the binding pair is added to capture said ligated products.
6. The method of claim 1 , wherein said 5′ ligation moiety is a thioester and said 3′ ligation moiety is a nucleophile.
7. The method of claim 1 , wherein said 5′ ligation moiety is a nucleophile and said 3′ ligation moiety is a thioester.
8. The method of claim 1 , wherein said 5′ ligation moiety is a DABSYL moiety and said 3′ ligation moiety is a phosphorothioate moiety.
9. The method of claim 1 , wherein said 5′ ligation moiety is a phosphorothioate moiety and said 3′ ligation moiety is a DABSYL moiety.
10. A method according to claim 1 wherein said guanidinium salt is guanidinium hydrochloride.
11. A method according to claim 1 wherein said guanidinium salt is guanidinium isothiocyanate.
12. A method according to claim 1 wherein said guanidinium salt is 3M guanidinium hydrochloride.
13. A method according to claim 1 wherein said detectable label is a fluorophore.
14. A method according to claim 1 wherein said capture support comprises beads.
15. A method according to claim 14 wherein said beads are magnetic beads.
16. A method according to claim 15 wherein said binding partner pair comprises biotin and streptavidin.
17. A method according to claim 16 wherein said biotin is attached to one of said ligation probes.