- Document Number:
20160340710
- Appl. No:
14/998162
- Application Filed:
December 24, 2015
- نبذة مختصرة :
Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting the sample, in the presence of a polymerase and an endonuclease, with a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of a target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.
- Claim:
1. A method for detecting a target nucleic acid in a sample, said method comprising contacting said sample with: a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of said target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide a polymerase; and an endonuclease for a nicking reaction.
- Claim:
2. The method of claim 1, wherein said method is performed at a substantially constant temperature.
- Claim:
3. The method of claim 1, wherein said method is performed at a temperature of from about 20° C. to about 42° C.
- Claim:
4. The method of claim 1, wherein said polymerase has strand displacement activity.
- Claim:
5. The method of claim 1, wherein said polymerase is 3′ to 5′ exonuclease deficient, 5′ to 3′ exonuclease deficient, or both.
- Claim:
6. The method of claim 1 wherein said polymerase comprises a DNA polymerase selected from the group consisting of Klenow fragments of DNA polymerase I derived from E. coli, 5′ to 3′ exonuclease-deficient Bst DNA polymerases derived from Bacillus stearothermophilus, and 5′ to 3′ exonuclease-deficient Bca DNA polymerases derived from Bacillus caldotenax.
- Claim:
7. The method of claim 1 wherein said endonuclease is an enzyme selected from the group consisting of Nb.BbvCI, Nt.AlwI, Nt.BbvCI, and Nt.BsmAI.
- Claim:
8. The method of claim 1 wherein said target is a microRNA.
- Claim:
9. The method of claim 1 wherein said target nucleic acid originates from an infectious agent.
- Claim:
10. A composition for detecting a target nucleic acid in a sample, said composition comprising: a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of said target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.
- Claim:
11. The composition of claim 10, further comprising a polymerase and an endonuclease for a nicking reaction.
- Claim:
12. The composition of claim 11 wherein said polymerase has strand displacement activity.
- Claim:
13. The composition of claim 11 wherein said polymerase is 3′ to 5′ exonuclease deficient, 5′ to 3′ exonuclease deficient, or both.
- Claim:
14. The composition of claim 11 wherein said polymerase comprises a DNA polymerase selected from the group consisting of Klenow fragments of DNA polymerase I derived from E. coli, 5′ to 3′ exonuclease-deficient Bst DNA polymerases derived from Bacillus stearothermophilus, and 5′ to 3′ exonuclease-deficient Bca DNA polymerases derived from Bacillus caldotenax.
- Claim:
15. The composition of claim 11 wherein said endonuclease is an enzyme selected from the group consisting of Nb.BbvCI, Nt.AlwI, Nt.BbvCI, and Nt.BsmAI.
- Claim:
16. The composition of claim 10 wherein said target nucleic acid is a microRNA.
- Claim:
17. The composition of claim 10 wherein said target nucleic acid originates from an infectious agent.
- Claim:
18. A kit for detecting a target nucleic acid in a sample, said kit comprising: a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of said target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.
- Claim:
19.-27. (canceled)
- Claim:
28. A method for detecting a target nucleic acid in a sample, said method comprising contacting said sample with a sequence conversion DNA, n unique cascade sequence amplification DNAs, a polymerase, and an endonuclease for a nicking reaction.
- Claim:
29. The method of claim 28, wherein n is an integer between 1 and 10.
- Claim:
30.-33. (canceled)
- Claim:
34. The method of claim 1, wherein said third oligonucleotide is a mini-circle DNA, and wherein a signal DNA binds to said mini-circle DNA and primes rolling circle amplification.
- Claim:
35. The method of claim 28, wherein at least one unique cascade sequence amplification DNA is a mini-circle DNA, and wherein a signal DNA binds to said mini-circle DNA and primes rolling circle amplification.
- Claim:
36. (canceled)
- Claim:
37. (canceled)
- Claim:
38. The method of claim 1, further comprising detecting the presence or absence of at least one signal DNA generated by at least one signal generation sequence.
- Claim:
39. (canceled)
- Current International Class:
12
- الرقم المعرف:
edspap.20160340710
Processing Request
No Comments.