Target-dependent reactions using structure-bridging oligonucleotides
View Patent ↗The present invention relates to methods and compositions for analyzing nucleic acids. In particular, the present invention provides methods and compositions for the detection and characterization of nucleic acids and sequence changes. The methods of the present invention permit the detection and/or identification of genetic polymorphism such as those associated with human disease and permit the identification of pathogens (e.g., viral and bacterial strain identification).
1. A method, comprising:
a) providing:
i) target nucleic acid comprising first and second non-contiguous single-stranded regions separated by a secondary structure;
ii) a first oligonucleotide that hybridizes across said secondary structure;
iii) a second oligonucleotide capable of binding to a portion of said first non-contiguous single-stranded region; and
iv) a 5′ nuclease;
b) mixing said target nucleic acid, said first oligonucleotide, said second oligonucleotide, and said 5′ nuclease under conditions such that a cleavage structure is formed and either said first oligonucleotide or said second oligonucleotide is cleaved.
2. The method of claim 1 , wherein said secondary structure comprises a hairpin loop region.
3. The method of claim 1 , wherein said secondary structure comprises a bend region.
4. The method of claim 1 , wherein said secondary structure comprises a bulge region.
5. The method of claim 1 , wherein said secondary structure comprises a stem-loop region.
6. The method of claim 1 , wherein said 5′ nuclease is thermostable.
7. The method of claim 1 , wherein said target nucleic acid is DNA.
8. The method of claim 7 , wherein said DNA is viral DNA.
9. The method of claim 1 , wherein said target nucleic acid is RNA.
10. The method of claim 9 , wherein said RNA is viral RNA.
11. The method of claim 1 , further comprising:
c) detecting the cleavage of said cleavage structure.
12. The method of claim 11 , wherein said detecting the cleavage of said cleavage structure comprises detection of fluorescence energy transfer.
13. The method of claim 11 , wherein said detecting the cleavage of said cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.
14. The method of claim 1 , wherein said first oligonucleotide is attached to a solid support.
15. The method of claim 1 , wherein said second oligonucleotide is attached to a solid support.
16. A method, comprising:
a) providing:
i) target nucleic acid comprising first and second non-contiguous single-stranded regions separated by a secondary structure;
ii) a first oligonucleotide that hybridizes across said secondary structure, wherein said first oligonucleotide comprises a region complementary to said first non-contiguous single-stranded region of said target nucleic acid;
iii) a second oligonucleotide capable of binding to a portion of said second non-contiguous single-stranded region, wherein said second oligonucleotide comprises a 3′ portion and a 5′ portion, wherein said 5′ portion comprises a region complementary to said second non-contiguous single-stranded region of said target nucleic acid; and
iv) a 5′ nuclease;
b) mixing said target nucleic acid, said first oligonucleotide, said second oligonucleotide, and said 5′ nuclease under conditions such that said first oligonucleotide hybridizes with said first non-contiguous single-stranded region of said target nucleic acid and such that said 5′ portion of said second oligonucleotide hybridizes with said second non-contiguous single-stranded region of said target nucleic acid so as to form a cleavage structure, wherein said 5′ nuclease cleaves said cleavage structure.
17. The method of claim 16 , wherein said cleaving of said cleavage structure comprises cleavage of said first oligonucleotide.
18. The method of claim 16 , wherein said cleaving of said cleavage structure comprises cleavage of said second oligonucleotide.
19. The method of claim 16 , wherein said secondary structure comprises a hairpin loop region.
20. The method of claim 16 , wherein said secondary structure comprises a bend region.
21. The method of claim 16 , wherein said secondary structure comprises a bulge region.
22. The method of claim 16 , wherein said secondary structure comprises a stem-loop region.
23. The method of claim 16 , wherein said 5′ nuclease is thermostable.
24. The method of claim 16 , wherein said target nucleic acid is DNA.
25. The method of claim 24 , wherein said DNA is viral DNA.
26. The method of claim 16 , wherein said target nucleic acid is RNA.
27. The method of claim 26 , wherein said RNA is viral RNA.
28. The method of claim 16 , further comprising:
c) detecting the cleavage of said cleavage structure.
29. The method of claim 28 , wherein said detecting the cleavage of said cleavage structure comprises detection of fluorescence energy transfer.
30. The method of claim 28 , wherein said detecting the cleavage of said cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.
31. The method of claim 16 , wherein said first oligonucleotide is attached to a solid support.
32. The method of claim 16 , wherein said second oligonucleotide is attached to a solid support.