Methods of using FET labeled oligonucleotides that include a 3′→5′ exonuclease resistant quencher domain and compositions for practicing the same
View Patent ↗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.
1. A method for detecting the production of a primer extension product in a primer extension reaction mixture, said method comprising:
(a) producing a primer extension mixture comprising a template nucleic acid, a nucleic acid polymerase having 3′→5′ exonuclease activity, and a FET labeled oligonucleotide comprising a target-binding sequence, a fluorophore, a nucleic acid intercalator, and a quencher, wherein said nucleic acid intercalator and said quencher are covalently bonded to the 3′ end of said FET labeled oligonucleotide;
(b) subjecting said primer extension mixture to primer extension reaction conditions; and
(c) detecting a fluorescent signal from said FET labeled oligonucleotide in said mixture, whereby a change in said fluorescent signal is indicative of the presence of a primer extension product in said mixture.
2. The method according to claim 1 , wherein said primer extension reaction is a PCR amplification reaction.
3. The method according to claim 2 , wherein said method is a real-time method of monitoring said PCR amplification reaction.
4. The method according to claim 1 , wherein said quencher is a dark quencher.
5. The method according to claim 4 , wherein energy transfer occurs between said fluorophore and dark quencher of said FET labeled oligonucleotide probe upon fluorophore excitation when said FET labeled oligonucleotide is not hybridized to target nucleic acid.
6. The method according to claim 5 , wherein energy transfer does not occur between said fluorophore and dark quencher of said FET labeled oligonucleotide probe upon fluorophore excitation when said FET labeled oligonucleotide probe is hybridized to a target nucleic acid.
7. The method according to claim 4 , wherein said dark quencher is a Black Hole Quencher.
8. The method according to claim 1 , wherein said FET labeled oligonucleotide is a probe selected from the group consisting of: a TAQMAN probe, a SCORPION probe, a SUNRISE probe, a molecular beacon, a conformationally assisted probes, and an in situ hybridization probe.
9. The method according to claim 1 , wherein said nucleic acid intercalator comprises a polycyclic compound.
10. The method according to claim 9 , wherein said polycyclic compound comprises an aromatic ring.
11. The method according to claim 9 , wherein said polycyclic compound comprises at least three rings and not more than six rings.
12. The method according to claim 9 , wherein said polycyclic compound comprises at least three rings, wherein at least two of said rings are fused.
13. The method according to claim 12 , wherein said polycyclic compound is an acridine.
14. The method according to claim 1 , wherein said nucleic acid intercalator is covalently bonded to said oligonucleotide through at least one linker.
15. The method according to claim 1 , wherein said nucleic acid intercalator is covalently bonded to the 3′ terminus of said oligonucleotide.
16. The method according to claim 15 , wherein said quencher is bonded to said nucleic acid intercalator.
17. The method according to claim 15 , wherein said quencher is bonded to the 3′ terminal nucleotide of the oligonucleotide.
18. The method according to claim 1 , wherein said quencher is covalently bonded to the 3′ terminus of said oligonucleotide.