Abasic site endonuclease assay
The present invention provides a novel method for detection and/or genotyping of nucleic acids that utilizes the specificity of an AP endonuclease. In addition, the present invention provides a novel method for nucleic acid amplification.
1. A kit comprising an AP site probe comprising an oligonucleotide NA that hybridizes to a target nucleic acid, a quencher molecule attached to the NA, and a functional tail R comprising a detectable reporter group, said functional tail R attached via a hydroxyprolinol linker, which is attached via a phosphodiester bond of a phosphate group to the 3′ terminal nucleotide of the NA, wherein the reporter group is not detected when the functional tail R is attached to the NA.
2. The kit of claim 1 , wherein the NA of said AP site probe is 3-200 nucleotides in length.
3. The kit of claim 1 , wherein the NA of said AP site probe is 3-100 nucleotides in length.
4. The kit of claim 1 , wherein the NA of said AP site probe is 5-30 nucleotides in length.
5. The kit of claim 1 , wherein said AP site probe comprises at least one modified base.
6. The kit of claim 1 , wherein the reporter group is a fluorophore.
7. The kit of claim 1 , further comprising an enhancer oligonucleotide, wherein the 5′-end of said enhancer oligonucleotide hybridizes to the target nucleic acid on the 3′ side of the hybridized AP site probe, wherein a gap of 1, 2 or 5 unpaired bases resides between the enhancer oligonucleotide and the AP site probe hybridization locations with the target nucleic acid.
8. The kit of claim 1 , wherein the quencher molecule is attached to the 5′ end of the NA of said AP site probe via a non-cleavable linker.
9. The kit of claim 7 , wherein said AP site probe is covalently linked to the 3′ end of said enhancer.
10. The kit of claim 7 , wherein said enhancer is 3 to 50 nucleotides in length.
11. The kit of claim 7 , wherein said enhancer is 5 to 30 nucleotides in length.
12. The kit of claim 1 , further comprising an AP endonuclease.
13. The kit of claim 12 , wherein the AP endonuclease is a Class II AP endonuclease.
14. The kit of claim 13 , wherein the Class II AP endonuclease is an E. coli Endonuclease IV.
15. The kit of claim 1 , wherein said target nucleic acid is a product of an amplification reaction.
16. The kit of claim 15 , wherein said amplification reaction is polymerase chain reaction.
17. The kit of claim 16 , wherein said polymerase chain reaction uses a thermostable endonuclease.
18. The kit of claim 15 , wherein said amplification reaction is an isothermal amplification reaction.
19. The kit of claim 1 , further comprising a second AP site probe, wherein said first probe comprises a NA portion comprising at least one base difference from the NA portion of said second probe, and wherein said first probe comprises a reporter group that is distinguishably detectable from the reporter group of said second probe.
20. The kit of claim 19 , wherein the reporter group of said first probe and said second probe comprises a fluorophore, and wherein the fluorophore of said first probe comprises a distinguishably detectable emission wavelength from the fluorophore of said second probe.
21. The kit of claim 19 , wherein said at least one base difference between the NA of said first probe and the NA of said second probe comprises a base difference at position 1, 2, 3 or 4 from the 3′ end of said probes.
22. The kit of claim 19 , wherein said at least one base difference between the NA of said first probe and the NA of said second probe comprises a base difference at position 1 or 2 from the 3′ end of said probes.
23. The kit of claim 1 , further comprising a plurality of AP site probes, wherein the NA portion of said probes are members of a universal library of about 5-8 nucleotides in length.
24. The kit of claim 23 , wherein each AP site probe member comprises at least one modified base.
25. The kit of claim 24 , wherein the modified base is selected from the group consisting of 5-hydroxybutynyl uridine; 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol; 4-amino-1H-pyrazolo[3,4-d]pyrimidine; 5-(4-Hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione; 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one; and 4-hydroxy-6-amino pyrazolopyrimidine.
26. The kit of claim 24 , wherein the modified base is selected from the group consisting of a phosphothioate, a methylphosphonate, a sulfamate, a polyamide and a locked nucleic acid.