Nucleic acid detection compositions
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 composition for detecting a target nucleic acid, comprising:
a) a purified thermostable FEN-1 endonuclease, active at 55° C. or above;
b) an oligonucleotide probe, said oligonucleotide probe configured to participate in a FEN-1 endonuclease cleavage structure in the presence of said target nucleic molecule.
2. The composition of claim 1 , wherein said oligonucleotide probe comprises a label.
3. The composition of claim 2 , wherein said label is configured to be detectable by a method comprising detection of fluorescence.
4. The composition of claim 2 , wherein said label is configured to be detectable by a method comprising detection of mass.
5. The composition of claim 2 , wherein said label is configured to be detectable by a method comprising detection of fluorescence energy transfer.
6. The composition of claim 2 , wherein said label is configured to be detectable by a method comprising detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, fluorescence polarization, and charge.
7. The composition of claim 1 , wherein said thermostable FEN-1 endonuclease is from an archaebacterium.
8. The composition of claim 7 , wherein said archaebacterium is Pyrococcus furiosus.
9. The composition of claim 7 , wherein said archaebacterium is Methanococcus jannaschii.
10. The composition of claim 1 , further comprising a nucleic acid molecule configured to hybridize to said target nucleic acid upstream of said oligonucleotide probe.
11. The composition of claim 10 , wherein said oligonucleotide probe is configured to hybridize to a first region of said target nucleic acid, and wherein said nucleic acid molecule comprises a 5′ portion and a 3′ portion, said 5′ portion being configured to hybridize to a second region of said target nucleic acid, wherein said second region of said target nucleic acid is downstream of and contiguous to said first region of said target nucleic acid.
12. The composition of claim 11 , wherein said 3′ portion of said nucleic acid molecule comprises an aromatic ring.
13. The composition of claim 11 , wherein said 3′ portion of said nucleic acid molecule comprises a 3′ terminal nucleotide not complementary to said target nucleic acid.
14. The composition of claim 11 , wherein said 3′ portion of said nucleic acid molecule consists of a single nucleotide.
15. The composition of claim 14 , wherein said single nucleotide is not complementary to said target nucleic acid.
16. The composition of claim 14 , wherein said single nucleotide is complementary to said target nucleic acid.
17. The composition of claim 13 , wherein said 3′ terminal nucleotide comprises a naturally occurring nucleotide.
18. The composition of claim 13 , wherein said 3′ terminal nucleotide comprises a nucleotide analog.
19. The composition of claim 11 , wherein said 3′ portion of said nucleic acid molecule is completely complementary to said target nucleic acid.
20. The composition of claim 1 , wherein said oligonucleotide probe is attached to a solid support.
21. The composition of claim 10 , wherein said nucleic acid molecule is attached to a solid support.
22. The composition of claim 1 , further comprising a polymerase enzyme.
23. The composition of claim 22 , wherein said polymerase enzyme is altered to have reduced synthetic activity.
24. The composition of claim 1 , further comprising said target nucleic acid.
25. The composition of claim 24 , wherein said target nucleic acid comprises DNA.
26. The composition of claim 24 , wherein said target nucleic acid comprises synthetic nucleic acid.
27. The composition of claim 26 , wherein said synthetic nucleic acid comprises an amplified nucleic acid.
28. The composition of claim 27 , wherein said amplified nucleic acid is produced using a polymerase chain reaction.
29. The composition of claim 11 , wherein said composition further comprises a stacker oligonucleotide, wherein said stacker oligonucleotide is configured to hybridize to a third region of said target nucleic acid, and wherein said third region of said target nucleic acid is upstream of and contiguous to said first region of said target nucleic acid.