Methods of detecting an amplified nucleic acid
View Patent ↗The present invention is directed to methods of generating a signal indicative of the presence of said target nucleic acid sequence in a sample, comprising, incubating a sample comprising a an amplified target nucleic acid and a nucleic acid polymerase which substantially lacks 5′ to 3′ exonuclease activity, adding a thermostable fen nuclease consisting of 5′ to 3′ exonuclease and/or endonuclease activity so as to cleave a cleavage structure and generate a signal.
1. A method of amplifying a target nucleic acid and generating a signal indicative of the presence of said target nucleic acid in a sample, comprising:
in a composition comprising an amplified target nucleic acid and a nucleic acid polymerase which substantially lacks 5′ to 3′ exonuclease activity, adding a thermostable fen nuclease consisting of 5′ to 3′ exonuclease and/or endonuclease activity so as to cleave a cleavage structure and generate a signal, said cleavage structure comprising duplex nucleic acid comprising an amplified target nucleic acid strand duplexed to an oligonucleotide comprising a single-stranded 5′ flap, wherein generation of said signal is indicative of the presence of a target nucleic acid in said sample.
2. The method of claim 1 , wherein said nucleic acid polymerase is thermostable.
3. The method of claim 1 or 2 , said composition comprising an amplification primer which produces an amplified target nucleic acid.
4. The method of claim 1 or 2 , said composition comprising one or more dNTPs.
5. The method of claim 2 , wherein said nucleic acid polymerase is selected from the group consisting of Pfu, Pfu-exo, Taq, Taq-exo, Deep vent and Deep vent exo.
6. The method of claim 1 or 2 , wherein said fen nuclease is selected from the group consisting of fen nuclease enzyme of Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus , and Pyrococcus horikoshii.
7. The method of claim 1 or 2 , wherein said 5′ flap is labeled.
8. The method of claim 7 , wherein said 5′ flap comprises a label comprising a first member of a pair of interactive labels effectively positioned to quench the generation of a detectable signal, the first and second members of said pair being separated by a site susceptible to fen nuclease cleavage, thereby allowing the nuclease activity of the fen nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moeity by cleaving at said site susceptible to fen nuclease, thereby generating a signal.
9. The method of claim 8 , wherein said pair of interactive labels comprises a quencher moiety and a fluorescent moiety.
10. A method of amplifying a target nucleic acid and generating a signal indicative of the presence of said target nucleic acid in a sample, comprising:
in a composition, forming a cleavage structure comprising duplex nucleic acid and a 5′ single-stranded nucleic acid flap by amplifying a target nucleic acid in the presence of a nucleic acid polymerase and a primer specific for said target nucleic acid under conditions which are permissive for nucleic acid amplification, and, in the presence of a thermostable fen nuclease consisting of 5′ to 3′ exonuclease and/or endonuclease activity, permitting cleavage of said cleavage structure to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid in said sample.
11. The method of claim 10 , wherein said composition comprises one or more dNTPs.
12. The method of claim 10 , wherein said nucleic acid polymerase substantially lacks 5′ to 3′ exonuclease activity.
13. The method of claims 10 - 12 , wherein said nucleic acid polymerase is thermostable.
14. The method of claim 13 , wherein said nucleic acid polymerase is selected from the group consisting of Pfu, Pfu-exo, Taq, Taq-exo, Deep vent and Deep vent exo.
15. The method of claim 10 , wherein said fen nuclease is selected from the group consisting of fen nuclease enzyme of Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus , and Pyrococcus horikoshii.
16. The method of claim 13 , wherein said 5′ flap is labeled.
17. The method of claim 16 , wherein said 5′ flap comprises a label comprising a first member of a pair of interactive labels effectively positioned to quench the generation of a detectable signal, the first and second members of said pair being separated by a site susceptible to fen nuclease cleavage, thereby allowing the nuclease activity of the fen nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moeity by cleaving at said site susceptible to fen nuclease, thereby generating a signal.
18. The method of claim 17 , wherein said pair of interactive labels comprises a quencher moiety and a fluorescent moiety.
19. The method of claim 3 , wherein said primer is between about 10 to 100 nucleotides in length.
20. The method of claim 3 , wherein said primer is between about 17-50 nucleotides in length.
21. The method of claim 3 , wherein said primer is between about 17-45 nucleotides in length.
22. The method of claim 10 , wherein said primer is between about 10 to 100 nucleotides in length.
23. The method of claim 10 , wherein said primer is between about 17-50 nucleotides in length.
24. The method of claim 10 , wherein said primer is between about 17-45 nucleotides in length.
25. The method of claim 1 , wherein said Fen nuclease is a Fen-1 nuclease.
26. The method of claim 10 , wherein said Fen nuclease is a Fen-1 nuclease.