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 a target nucleic acid comprising:
exposing a composition comprising a purified thermostable FEN-1 endonuclease from an archaebacterial species selected from the group consisting of Pyrococcus furiosus and Methanococcus jannaschii and a purified nucleic acid molecule, wherein said purified nucleic acid molecule is capable of hybridizing to a target nucleic acid to form a FEN-1 endonuclease cleavage structure, to a sample suspected of containing said target nucleic acid;
forming a FEN-1 endonuclease cleavage structure; and
cleaving said FEN-1 endonuclease cleavage structure with said purified thermostable FEN-1 endonuclease, such that said target nucleic acid is detected.
2. The method of claim 1 , wherein said composition further comprises a polymerase, wherein said polymerase synthesizes a copy of said target nucleic acid or synthesizes a detectable product indicative of the cleavage of said FEN-1 endonuclease cleavage structure.
3. The method of claim 2 , wherein said polymerase comprises a thermostable polymerase.
4. The method of claim 2 , wherein said polymerase comprises a template-independent polymerase.
5. The method of claim 2 , wherein said polymerase comprises a template-dependent polymerase.
6. The method of claim 1 , wherein said purified thermostable FEN-1 endonuclease and said purified nucleic acid molecule are in a mixture.
7. The method of claim 1 , further comprising a second nucleic acid molecule capable of hybridizing to said FEN-1 endonuclease cleavage structure.
8. The method of claim 7 , wherein said second nucleic acid molecule comprises a probe oligonucleotide.
9. The method of claim 7 , wherein said second nucleic acid molecule is attached to a solid support.
10. The method of claim 7 , wherein said second nucleic acid molecule comprises a label.
11. The method of claim 10 , wherein said label comprises a fluorescent label.
12. The method of claim 7 , wherein said purified nucleic acid molecule and said second nucleic acid molecule are capable of hybridizing to a target nucleic acid having a first region and a second region, said second region downstream of and contiguous to said first region, wherein at least a portion of said purified nucleic acid molecule is completely complementary to said first region of said target nucleic acid and wherein said second nucleic acid molecule comprises a 3″ portion and a 5′ portion, wherein said 5′ portion is completely complementary to said second region of said target nucleic acid.
13. The method of claim 12 , further comprising hybridizing a stacker oligonucleotide to said target nucleic acid.
14. The method of claim 1 , wherein said detecting comprises detecting a non-target cleavage product.
15. The method of claim 12 , wherein said 3′ portion of said second nucleic acid molecule consists of a single nucleotide not complementary to said target nucleic acid.
16. The method of claim 12 , wherein said 3′ portion of said second nucleic acid molecule comprises a 3′ terminal nucleotide not complementary to said target nucleic acid.
17. The method of claim 12 , wherein said 3′ portion of said second nucleic acid molecule comprises a 3′ terminal nucleotide complementary to said target nucleic acid.
18. The method of claim 12 , wherein said 3′ portion of said second nucleic acid molecule comprises a single nucleotide.
19. The method of claim 12 , wherein a 3′ terminus of said 3′ portion of said second nucleic acid molecule comprises an aromatic ring structure that is not a base.
20. The method of claim 12 , wherein said purified nucleic acid molecule further comprises a 5′ terminal portion that is not complementary to said target nucleic acid.
21. The method of claim 1 , wherein said detecting comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, and fluorescence polarization.
22. The method of claim 14 , wherein said detecting comprises detection based on the charge of said non-target cleavage product.
23. The method of claim 1 , wherein said target nucleic acid is DNA.
24. The method of claim 1 , wherein said target nucleic acid is synthetic nucleic acid.
25. The method of claim 24 , wherein said synthetic nucleic acid comprises amplified nucleic acid.
26. The method of claim 25 , wherein said synthetic nucleic acid is generated by a polymerase chain reaction.
27. The method of claim 1 , wherein said target nucleic acid is RNA.