Kits for detection of nucleic acids
View Patent ↗The present invention relates to compositions and methods for the detection and characterization of small nucleic acid molecules (e.g., RNA (e.g., small RNAs such as micro RNAs (miRNAs) and small interfering RNAs (siRNAs)) and other short nucleic acid molecules). More particularly, the present invention relates to methods for the detection and quantification of RNA expression. The present invention further provides for the detection of miRNA and siRNA variants.
1. A kit for detecting an miRNA target comprising a first region, a second region, and a third region, wherein said first region is downstream of and contiguous to said second region, and wherein said second region is downstream of and contiguous to said third region, comprising:
i) a first oligonucleotide having a 3′ end, wherein said first oligonucleotide comprises a 3′ portion that is complementary to said first region of said miRNA target, and a 5′ portion that is not complementary to said miRNA target, wherein said 3′ portion of said first oligonucleotide comprises the 3′ end of said first oligonucleotide;
ii) a second oligonucleotide having a 3′ end, wherein said second oligonucleotide comprises a 3′ portion that is substantially homologous to said third region of said miRNA target and a 5′ portion that is not homologous to said third region of said miRNA target, wherein said 3′ portion of said second oligonucleotide comprises the 3′ end of said second oligonucleotide;
iii) a reverse transcriptase;
iv) a DNA polymerase;
v) a 5′ nuclease;
vi) a probe oligonucleotide, wherein at least a portion of said probe oligonucleotide is complementary to at least a portion of said second region of said miRNA target; and
vii) a first stacker oligonucleotide comprising a 5′ terminal portion, wherein at least the 5′ terminal portion of said stacker oligonucleotide is complementary to a region in said 5′ portion of said first oligonucleotide adjacent to said 3′ portion of said first oligonucleotide.
2. The kit of claim 1 , wherein said 5′ nuclease is thermostable.
3. The kit of claim 1 , wherein said probe oligonucleotide is unlabeled and contains a 5′ and 3′ region, wherein said 3′ region is complementary to said second region of said miRNA target; and wherein said 5′ region is non-complementary to said miRNA target and forms a 5′ flap.
4. The kit of claim 1 , wherein said 3′ portion of said first oligonucleotide is configured to form a duplex of about 6-10 base pairs with said miRNA.
5. The kit of claim 1 , further comprising a second stacker oligonucleotide comprising a 5′ terminal portion, wherein at least the 5′ terminal portion of said second stacker oligonucleotide is complementary to a region of said 5′ portion said second oligonucleotide adjacent to said 3′ portion of said second oligonucleotide.
6. The kit of claim 1 , wherein said probe is a labeled probe configured for FRET detection.
7. The kit of claim 1 , wherein said miRNA is selected from the group consisting of Let-7, miR-1, miR-135, miR-15, miR-16, miR125b, miR-1d, and miR124a.
8. The kit of claim 1 , wherein said 5′ nuclease comprises a modified DNA polymerase, wherein said modified DNA polymerase has 5′ nuclease activity but lacks synthetic activity.
9. The kit of claim 3 , further comprising an oligonucleotide cassette, wherein a portion of said oligonucleotide cassette is complementary to said 5′ region of said probe oligonucleotide, wherein said oligonucleotide cassette is configured to form an invasive cleavage structure with said non-target cleavage product from said cleavage of said probe oligonucleotide.
10. The kit of claim 9 , wherein said oligonucleotide cassette is configured for FRET detection.
11. The kit of claim 1 , wherein said 5′ nuclease is a FEN-1 endonuclease.
12. The kit of claim 11 , wherein said FEN-1 endonuclease comprises a thermostable FEN-1.
13. The kit of claim 12 , wherein said FEN-1 endonuclease is from an archaebacterial species.
14. The kit of claim 13 , wherein said archaebacterial species is selected from the group consisting of: Pyrococcus furiosus, Methanococcus jannaschii, Archaeoglobus veneficus and Archaeoglobus fulgidus.