Nucleic acid detection kits
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. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.
1. A kit comprising:
a) a first oligonucleotide comprising a 3′ portion and a 5′ portion said 3′ portion completely complementary to a first region of a target nucleic acid wherein said 3′ portion when hybridized to said target nucleic acid forms a hybridized region and said 5′ portion non-complementary to said target nucleic acid; and
b) a second oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′ portion completely complementary to a second region of said target nucleic acid downstream of and contiguous to said first region wherein said 3′ portion extends into said hybridized region; and
c) a structure-specific 5′-nuclease.
2. The kit of claim 1 , wherein said non-complementary 5′ portion of said first oligonucleotide is less than 10 nucleotides in length.
3. The kit of claim 1 , wherein said non-complementary 5′ portion of said first oligonucleotide is between 10 and 20 nucleotides in length.
4. The kit of claim 1 , wherein said non-complementary 5′ portion of said first oligonucleotide is greater than 20 nucleotides in length.
5. The kit of claim 1 , wherein said first and second regions of said target nucleic acid are longer than one nucleotide in length.
6. The kit of claim 1 , wherein said 3′ portion of said second oligonucleotide comprises a 3′ terminal nucleotide not complementary to said target nucleic acid.
7. The kit of claim 1 , wherein said 3′ portion of said second oligonucleotide consists of a single nucleotide not complementary to said target nucleic acid.
8. The kit of claim 1 , wherein said structure-specific 5′-nuclease comprises a thermostable structure-specific 5′-nuclease.
9. The kit of claim 8 , wherein said thermostable structure-specific 5′-nuclease comprises a Flap-endonuclease.
10. The kit of claim 9 , wherein said Flap-endonuclease comprises a FEN-1 endonuclease.
11. The kit of claim 1 , further comprising a polymerase.
12. The kit of claim 1 , further comprising a ligase.
13. The kit of claim 1 , wherein a portion of said structure-specific 5′-nuclease is homologous to a portion of a thermostable DNA polymerase derived from a thermophilic organism.
14. The kit of claim 13 , wherein said thermophilic organism is selected from the group consisting of Thermus aquaticus, Thermus flavus , and Thermus thermophilus.
15. The kit of claim 1 , wherein said kit further comprises a solid support.
16. The kit of claim 15 , wherein said first oligonucleotide is attached to said solid support.
17. The kit of claim 15 , wherein said second oligonucleotide is attached to said solid support.
18. The kit of claim 1 , further comprising a buffer solution.
19. The kit of claim 18 , wherein said buffer solution comprises a source of divalent cations.
20. The kit of claim 19 , wherein said divalent cation is selected from the group consisting of Mn 2+ and Mg 2+ ions.
21. The kit of claim 1 , further comprising a third oligonucleotide complementary to a third portion of said target nucleic acid upstream of said first portion of said target nucleic acid.
22. The kit of claim 1 , further comprising said target nucleic acid.
23. The kit of claim 1 , further comprising a second target nucleic acid.
24. The kit of claim 23 , further comprising a third oligonucleotide comprising a 5′ portion complementary to a first region of said second target nucleic acid.
25. The kit of claim 24 , wherein said 3′ portion of said third oligonucleotide is covalently linked to said second target nucleic acid.
26. The kit of claim 24 , wherein said second target nucleic acid further comprises a 5′ portion, wherein said 5′ portion of said second target nucleic acid is said third oligonucleotide.
27. The kit of claim 1 , further comprising a competitor oligonucleotide configured to bind said first oligonucleotide, and not bind said second oligonucleotide.