RNA detection assays
View Patent ↗The present invention provides novel cleavage agents and polymerases for the cleavage and modification of nucleic acid. The cleavage agents and polymerases find use, for example, for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. In some embodiments, the 5′ nuclease activity of a variety of enzymes is used to cleave a 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 comprising:
a) forming an invasive cleavage structure, said invasive cleavage structure comprising an RNA target nucleic acid;
b) cleaving said invasive cleavage structure with a cleavage agent comprising an isolated composition comprising the polypeptide of SEQ ID NO:2668 to produce a non-target cleavage product; and
c) detecting the cleavage of said invasive cleavage structure,
wherein said target nucleic acid comprises a first region and a second region, said second region downstream of and contiguous to said first region, and wherein said invasive cleavage structure comprises said target nucleic acid, a first oligonucleotide, and a second oligonucleotide, wherein at least a portion of said first oligonucleotide is completely complementary to said first region of said first target nucleic acid, and wherein said second oligonucleotide comprises a 3′ portion and a 5′ portion, wherein said 5′ portion is completely complementary to said second region of said target nucleic acid.
2. The method of claim 1 , wherein at least said portion of said first oligonucleotide is annealed to said first region of said target nucleic acid and wherein at least said 5′ portion of said second oligonucleotide is annealed to said second region of said target nucleic.
3. The method of claim 2 , wherein said 3′ portion of said second oligonucleotide comprises a 3′ terminal nucleotide not complementary to said target nucleic acid.
4. The method of claim 3 , wherein said 3′ portion of said second oligonucleotide consists of a single nucleotide not complementary to said target nucleic acid.
5. The method of claim 1 , wherein said detecting the cleavage of said invasive cleavage structure comprises detection of fluorescence.
6. The method of claim 1 , wherein said detecting the cleavage of said invasive cleavage structure comprises detection of mass.
7. The method of claim 1 , wherein said detecting the cleavage of said invasive cleavage structure comprises detection of fluorescence energy transfer.
8. 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, fluorescence polarization, and charge.
9. The method of claim 1 , further comprising the steps of forming a second invasive cleavage structure comprising said non-target cleavage product and cleaving said second invasive cleavage structure.
10. The method of claim 1 , wherein said RNA target nucleic acid comprises a cytochrome P450 RNA.
11. The method of claim 1 , wherein said RNA target nucleic acid comprises a cytokine RNA.
12. The method of claim 1 , wherein a portion of said target nucleic acid consisting of said first region and said second region of said target nucleic acid comprises a splice junction.
13. The method of claim 1 , wherein said first region or said second region of said target nucleic acid comprises an exon.
14. The method of claim 1 , wherein said first region or said second region of said target nucleic acid comprises an intron.
15. The method of claim 1 , wherein said RNA target nucleic acid is provided in a cell lysate.
16. The method of claim 1 , wherein said first oligonucleotide is covalently attached to said second oligonucleotide.
17. A method for detecting the presence of two or more target nucleic acid sequences comprising:
a) cleaving two or more invasive cleavage structures with a cleavage agent comprising an isolated composition comprising the polypeptide of SEQ ID NO:2668, each of said two or more invasive cleavage structures comprising an RNA target nucleic acid having a target sequence, wherein each of said invasive cleavage structures comprises a different RNA target sequence; and
b) detecting the cleavage of said invasive cleavage structure.
18. A recombinant nucleic acid composition comprising a nucleic acid encoding the polypeptide of SEQ ID NO:2668.
19. A vector comprising the nucleic acid sequence of claim 18 .
20. A host cell comprising the vector of claim 19 .
21. The composition of claim 18 , wherein said nucleic acid comprises SEQ ID NO:2702.
22. An isolated composition comprising the polypeptide of SEQ ID NO:2668.