Cleavage of nucleic acids
View Patent ↗The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.
1. A method for detecting the presence of a target nucleic acid molecule comprising:
a) forming a cleavage structure between a target nucleic acid, a first oligonucleotide, and a second oligonucleotide, wherein the target nucleic acid comprises a first region, a second region, and a third region, said first region downstream of and contiguous to said second region, and said second region downstream of and contiguous to said third region, wherein said first oligonucleotide comprises a 3′ portion and a 5′ portion, wherein said 3′ portion of said first oligonucleotide contains a sequence of one or more nucleotides that is completely complementary to said third region of said target nucleic acid, and wherein said 5′ portion of said first oligonucleotide contains a sequence of one or more nucleotides contiguous to said 3′ portion of said first oligonucleotide that is completely complementary to said second region of said target nucleic acid, wherein said second oligonucleotide comprises a 3′ portion and a 5′ portion, wherein said 5′ portion contains a sequence or one or more nucleotides that are completely complementary to said first region of said target nucleic acid, wherein said cleavage structure is formed when said first oligonucleotide is annealed to said third and said second region of said target nucleic acid and wherein said second oligonucleotide is annealed to said first region of said target nucleic acid such that the 3′ portion of said second oligonucleotide overlaps with the sequence of nucleotides in said 5′ portion of said first oligonucleotide annealed to said second region of said target nucleic acid;
b) cleaving said cleavage structure with a thermostable enzyme having 5′ nuclease activity and substantially lacking nucleic acid polymerase activity to generate a non-target cleavage product, and
c) detecting the cleavage of said cleavage structure to detect the presence of the target nucleic acid molecule.
2. The method of claim 1 , wherein said detecting the cleavage of cleavage structure comprises detecting said non-target cleavage product.
3. The method of claim 1 , wherein said 3′ portion of said second oligonucleotide is complementary to said second region of said target nucleic acid.
4. The method of claim 1 , wherein a 3′ terminal nucleotide of said 3′ portion of said second oligonucleotide comprises a nucleotide that is mismatched to the nucleotide at the corresponding position in said target nucleic acid.
5. The method of claim 1 , wherein said 3′ portion of said second oligonucleotide consists of a single nucleotide, said second region of said target nucleic acid consists of a single nucleotide, and wherein said 3′ portion of said second oligonucleotide is mismatched with said second region of said target nucleic acid.
6. The method of claim 3 , wherein said 3′ portion of said second oligonucleotide consists of a single nucleotide, said second region of said target nucleic acid consists of a single nucleotide, and wherein said 3′ portion of said second oligonucleotide is complementary to said second region of said target nucleic acid.
7. The method of claim 1 , wherein said a 3′ terminus of said 3′ portion of said second oligonucleotide comprises an aromatic ring structure that is not a base.
8. The method of claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection of fluorescence.
9. The method of claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection of mass.
10. The method of claim 1 , wherein the said cleavage structure is labeled with fluorophore and with a component that can suppress emission in combination with said fluorophore by fluorescence energy transfer, wherein said detecting the cleavage of said cleavage structure comprises detection of a change in fluorescence energy transfer through cleavage.
11. The method of claim 10 , wherein said component that can suppress emission in combination with said fluorophore by fluorescence energy transfer comprises a second fluorophore.
12. The method of claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, and fluorescence polarization.
13. The method of claim 1 , wherein said detecting the cleavage of said cleavage structure comprises detection based on the charge of non-target cleavage product.
14. The method of claim 1 , wherein said thermostable enzyme comprises an amino acid sequence, where in a portion of the amino acid sequence of said thermostable enzyme is homologous to a portion of an amino acid sequence of a thermostable DNA polymerase derived a Thermus species.
15. The method of claim 1 , wherein said thermostable enzyme is a FEN-1 nuclease.
16. The method of claim 15 , wherein said FEN-1 nuclease is from an archeabacterium.
17. The method of claim 16 , wherein said archaebacterium is Methanococcus jannaschii.
18. The method of claim 16 , wherein said archaebacterium is Pyrococcus woesei.
19. The method of claim 16 , wherein said archaebacterium is Pyrococcus furiosus.
20. The method of claim 1 , wherein said target nucleic acid is DNA.
21. The method of claim 1 wherein said target nucleic acid is synthetic nucleic acid.
22. The method of claim 21 , wherein said synthetic nucleic acid comprises amplified nucleic acid.
23. The method of claim 22 , wherein said amplified nucleic acid is generated by a polymerase chain reaction.
24. The method of claim 1 , wherein said target nucleic acid is RNA.
25. The method of claim 1 , wherein said first oligonucleotide further comprises a 5′ terminal portion that is not complementary to said target nucleic acid.
26. The method of claim 1 , wherein one or more of said first and said second oligonucleotides are generated by extension of a primer with template-dependent DNA polymerase.
27. The method of claim 1 , further comprising a third oligonucleotide complementary to a fourth region of said target nucleic acid, wherein said third region of said target nucleic acid is adjacent to and downstream of said fourth region of said target nucleic acid, and wherein said forming of said cleavage structure comprises annealing said third oligonucleotide to said fourth region of said target nucleic acid.