IP Library › Granted Patent US 8,822,673
Granted Patent B2
US 8,822,673 · App. 12/945,777 · Granted Sep 2, 2014

Methods of using FET labeled oligonucleotides that include a 3′-5′ exonuclease resistant quencher domain and compositions for practicing the same

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Quick Facts
Patent No.
US 8,822,673
App. No.
12/945,777
Granted
Sep 2, 2014
Kind
B2
Abstract

Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.

Claims (20)

1. A FET labeled probe comprising a polycyclic nucleic acid intercalator bonded to a FET labeled oligonucleotide, wherein the oligonucleotide comprises a single-stranded target-binding sequence linked to a fluorophore and a dark quencher, and wherein the intercalator and the dark quencher are covalently bonded to the 3′ terminus of the oligonucleotide.

2. The FET labeled probe according to claim 1 , wherein the polycyclic nucleic acid intercalator comprises an aromatic ring.

3. The FET labeled probe according to claim 1 , wherein the FET labeled oligonucleotide is selected from the group consisting of: a TAQMAN probe, a SCORPION probe, a SUNRISE probe, a molecular beacon, a conformationally assisted probe, and an in situ hybridization probe.

4. The FET labeled probe according to claim 1 , wherein the dark quencher is a Black Hole Quencher.

5. The FET labeled probe according to claim 1 , wherein the intercalator is covalently bonded to the oligonucleotide through at least one linker.

6. The FET labeled probe according to claim 1 , wherein the intercalator is directly bonded to the 3′ terminal nucleotide of the oligonucleotide.

7. The FET labeled probe according to claim 6 , wherein the quencher is bonded to the intercalator.

8. The FET labeled probe according to claim 6 , wherein the quencher is bonded to the 3′ terminal nucleotide of the oligonucleotide.

9. A FET labeled probe comprising a polycyclic nucleic acid intercalator bonded to a FET labeled oligonucleotide, wherein the oligonucleotide comprises a single-stranded target-binding sequence linked to a fluorophore and a quencher, wherein the intercalator and the quencher are covalently bonded to the 3′ terminus of the oligonucleotide, and wherein the oligonucleotide is selected from the group consisting of: a TAQMAN probe, a SCORPION probe, a SUNRISE probe, a molecular beacon, a conformationally assisted probe, and an in situ hybridization probe.

10. The FET labeled probe according to claim 9 , wherein the intercalator is covalently bonded to the oligonucleotide through at least one linker.

11. The FET labeled probe according to claim 9 , wherein the intercalator is directly bonded to the 3′ terminal nucleotide of the oligonucleotide.

12. The FET labeled probe according to claim 11 , wherein the quencher is bonded to the intercalator.

13. The FET labeled probe according to claim 11 , wherein the quencher is bonded to the 3′ terminal nucleotide of the oligonucleotide.

14. A probe comprising a polycyclic nucleic acid intercalator bonded to a FET labeled oligonucleotide, wherein the oligonucleotide comprises a single-stranded target-binding sequence linked to a fluorophore and a dark quencher, and wherein the intercalator and the quencher extend from the 3′ terminal nucleotide of the oligonucleotide.

15. The probe according to claim 14 , wherein the intercalator extends from the oligonucleotide through at least one linker.

16. The probe according to claim 14 , wherein the intercalator extends directly from the 3′ terminal nucleotide.

17. The probe according to claim 16 , wherein the quencher extends from the intercalator.

18. The probe according to claim 14 , wherein the quencher extends from the oligonucleotide through at least one linker.

19. The probe according to claim 14 , wherein the quencher extends directly from the 3′ terminal nucleotide.

20. The probe according to claim 19 , wherein the intercalator extends from the quencher.