Compositions and kits for detection of a target nucleic acid
View Patent ↗The present invention is directed to kits and compositions for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising a nucleic acid polymerase lacking 5′ to 3′ exonuclease activity and a thermostable FEN nuclease consisting of a 5′ to 3′ exonuclease and/or endonuclease activity.
1. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising a thermostable Fen nuclease consisting of a 5′ to 3′ exonuclease and/or endonuclease activity, a nucleic acid polymerase that substantially lacks 5′ to 3′ exonuclease activity, a nucleic acid primer, and a labeled nucleic acid complementary to said target nucleic acid sequence.
2. The kit of claim 1 , comprising one or more dNTPs.
3. The kit of claim 1 or 2 , wherein said nucleic acid polymerase has less than 10% of the exonuclease activity of a wild type polymerase.
4. The kit of claim 3 , wherein said nucleic acid polymerase has less than 5% of the exonuclease activity of a wild type polymerase.
5. The kit of claim 4 , wherein said nucleic acid polymerase has less than 1% of the exonuclease activity of a wild type polymerase.
6. The kit of claim 1 or 2 , wherein said nucleic acid polymerase is thermostable.
7. The kit of claim 6 , wherein said Fen nuclease is selected from the group consisting of Fen nuclease enzyme of Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus , and Pyrococcus horikoshii.
8. The kit of claim 6 , wherein said nucleic acid polymerase is selected from the group consisting of Pfu, Pfu-exo, Taq, Taq-exo, Deep vent and Deep vent exo.
9. The kit of claim 1 or 2 , wherein said labeled complementary nucleic acid 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 separable 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 moiety by cleaving at said site susceptible to Fen nuclease, thereby generating a signal.
10. The kit of claim 9 , wherein said pair of interactive labels comprises a quencher moiety and a fluorescent moiety.
11. A composition comprising an isolated thermostable nucleic acid polymerase substantially lacking 5′ to 3′ exonuclease activity, a isolated thermostable Fen nuclease consisting of a 5′ to 3′ exonuclease and/or endonuclease activity, and a nucleic acid primer.
12. The composition of claim 11 , comprising one or more dNTPs.
13. The composition of claim 11 or 12 , wherein said Fen nuclease is selected from the group consisting of a Fen nuclease enzyme of Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus , and Pyrococcus horikoshii.
14. The composition of claim 11 or 12 , wherein said nucleic acid polymerase is selected from the group consisting of Pfu, Pfu-exo, Taq, Tag-exo, Deep vent and Deep vent exo.
15. The composition of claim 12 , wherein said nucleic acid polymerase has less than 10% of the exonuclease activity of a wild type polymerase.
16. The composition of claim 15 , wherein said nucleic acid polymerase has less than 5% of the exonuclease activity of a wild type polymerase.
17. The composition of claim 16 , wherein said nucleic acid polymerase has less than 1% of the exonuclease activity of a wild type polymerase.
18. The composition of claim 12 , further comprising a labeled nucleic acid complementary to a target nucleic acid sequence in a sample comprising a target nucleic acid.
19. The composition of claim 18 , wherein said labeled complementary nucleic acid 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 separable 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 moiety by cleaving at said site susceptible to Fen nuclease, thereby generating a signal.
20. The composition of claim 19 , wherein said pair of interactive labels comprises a quencher moiety and a fluorescent moiety,
21. The composition of claim 11 , 12 or 18 , further comprising said target nucleic acid.
22. The kit of claim 1 , wherein said nucleic acid primer is between about 10 to 100 nucleotides in length.
23. The kit of claim 1 , wherein said nucleic acid primer is between about 17-50 nucleotides in length.
24. The kit of claim 1 , wherein said nucleic acid primer is between about 17-45 nucleotides in length.
25. The composition of claim 11 , wherein said nucleic acid primer is between about 10 to 100 nucleotides in length.
26. The composition of claim 11 , wherein said nucleic acid primer is between about 17-50 nucleotides in length.
27. The composition of claim 11 , wherein said nucleic acid prime is between about 17-45 nucleotides in length.
28. The kit of claim 1 , wherein said Fen nuclease is a Fen-1 nuclease.
29. The composition of claim 11 , wherein said Fen nuclease is a Fen-1 nuclease.
30. The kit of claim 5 , wherein said nucleic acid polymerase is thermostable.
31. The kit of claim 6 , wherein said labeled complementary nucleic acid 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 separable 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 moiety by cleaving at said site susceptible to Fen nuclease, thereby generating a signal.
32. The kit of claim 30 , wherein said labeled complementary nucleic acid 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 separable 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 moiety by cleaving at said site susceptible to Fen nuclease, thereby generating a signal.