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 kit for detecting a target nucleic acid, comprising:
i) a purified thermostable FEN-1 endonuclease; and
ii) an oligonucleotide probe configured to hybridize to a target nucleic acid to form a FEN-1 endonuclease cleavage structure.
2. The kit of claim 1 , further comprising a nucleic acid molecule configured to hybridize to said target nucleic acid upstream of said oligonucleotide probe.
3. The kit of claim 2 , wherein said oligonucleotide probe comprises a label.
4. The kit of claim 2 , further comprising a stacker oligonucleotide configured to hybridize to said target nucleic acid adjacent to and downstream of said oligonucleotide probe.
5. The kit of claim 2 , wherein said purified FEN-1 endonuclease is from an archaebacterial species.
6. The kit of claim 5 , wherein said purified FEN-1 endonuclease is thermostable.
7. The kit of claim 2 , wherein said purified FEN-1 endonuclease is from an archaebacterial species selected from the group consisting of Pyrococcus furiosus and Methanococcus jannaschii.
8. The kit of claim 2 , further comprising a polymerase.
9. The kit of claim 7 , wherein said polymerase comprises a thermostable polymerase.
10. The kit of claim 8 , wherein said polymerase comprises a template-independent polymerase.
11. A kit for detecting a target sequence, comprising:
i) a purified thermostable FEN-1 endonuclease;
ii) a target nucleic acid, which comprises in 3′ to 5′ order a first region and a second region;
iii) a first oligonucleotide that is at least partially complementary to said first region of said target nucleic acid, and
iv) a second oligonucleotide comprising a 5′ portion and a 3′ portion, wherein said 3′ portion is complementary to said second region of said target nucleic acid and wherein said 5′ portion is not complementary to said target nucleic acid;
wherein said first and second oligonucleotides form an invasive cleavage structure when hybridized to said target nucleic acid.
12. The kit of claim 11 , wherein said 5′ portion of said second oligonucleotide comprises a label.
13. The kit of claim 11 , further comprising a stacker oligonucleotide configured to hybridize to said target nucleic acid adjacent to and downstream of said oligonucleotide probe.
14. The kit of claim 11 , wherein said purified FEN-1 endonuclease is from an archaebacterial species.
15. The kit of claim 14 , wherein said purified FEN-1 endonuclease is thermostable.
16. The kit of claim 14 , wherein said purified FEN-1 endonuclease is from an archaebacterial species selected from the group consisting of Pyrococcus furiosus and Methanococcus jannaschii.
17. The kit of claim 11 , further comprising a polymerase.
18. The kit of claim 17 , wherein said polymerase comprises a thermostable polymerase.
19. The kit of claim 17 , wherein said polymerase comprises a template-independent polymerase.
20. The kit of claim 17 , wherein said polymerase comprises a template-dependent polymerase.