METHODS OF USING FET LABELED OLIGONUCLEOTIDES THAT INCLUDE A 3′-5′ EXONUCLEASE RESISTANT QUENCHER DOMAIN AND COMPOSITIONS FOR PRACTICING THE SAME
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 - 28 . (canceled)
29 . A FET labeled probe comprising nucleic acid intercalator bonded to a FET labeled oligonucleotide.
30 . The FET labeled probe according to claim 29 , wherein said intercalator covalently bonded to said FET labeled oligonucleotide.
31 . The FET labeled probe according to claim 29 , wherein said nucleic acid intercalator is located at the 3 end of said FET labeled oligonucleotide.
32 . The FET labeled probe according to claim 29 , wherein said nucleic acid intercalator is located at the 5′ end of said FET labeled oligonucleotide.
33 . The FET labeled probe according to claim 29 , wherein said nucleic acid intercalator provides increased stability to the hybrid formed from said FET labeled oligonucleotide.
34 . The FET labeled probe according to claim 29 , wherein said nucleic acid intercalator provides exonuclease activity resistance to said FET labeled oligonucleotide.
35 . The FET labeled probe according to claim 29 , wherein said nucleic acid intercalator comprises a polycyclic compound.
36 . The FET labeled probe according to claim 35 , wherein said polycyclic compound comprises an aromatic ring.
37 . The FET labeled probe according to claim 35 , wherein said polycyclic compound comprises at least three rings and not more than six rings.
38 . The FET labeled probe according to claim 35 , wherein said polycyclic compound comprises at least three rings, wherein at least two of said rings are fused.
39 . The FET labeled probe according to claim 38 , wherein said polycyclic compound is an acridine.
40 . The FET labeled probe according to claim 29 , wherein said FET labeled oligonucleotide is a nucleic acid detector molecule that includes a single-stranded target binding sequence linked to fluorophore and dark quencher.
41 . The FET labeled probe according to claim 40 , wherein said FET labeled oligonucleotide is a probe selected from the group consisting of: Taqman probes, scorpion probes, sunrise probes, molecular beacons, conformationally assisted probes, and in situ hybridization probes.
42 . A method of monitoring of a PCR amplification reaction, said method comprising:
preparing a PCR amplification reaction mixture by combining:
a template nucleic acid;
forward and reverse nucleic acid primers;
deoxyribonucleotides;
a nucleic acid polymerase;
a FET labeled oligonucleotide that includes: a 3′→5′ exonuclease resistant quencher domain comprising a dark quencher, a fluorescent reporter domain comprising a fluorophore and a PCR product complementary domain; and
at least one of:
a nucleic acid intercalator, and
a minor groove binder;
subjecting said PCR amplification reaction mixture to PCR amplification conditions;
monitoring said reaction mixture for a fluorescent signal from said FET labeled oligonucleotide probe to obtain an assay result; and
employing said assay result to monitor said PCR amplification reaction.
43 . The method according to claim 42 , wherein said method is a method of monitoring a PCR amplification reaction in real time.
44 . The method according to claim 42 , wherein said FET labeled oligonucleotide is a probe selected from the group consisting of: scorpion probes, sunrise probes, molecular beacons, and conformationally assisted probes.
45 . A method for screening a nucleic acid sample for the presence of first and second nucleic acids that differ from each other by a single nucleotide, said method comprising:
producing a primer extension mixture that includes:
said nucleic acid sample;
a nucleic acid polymerase;
first and second FET labeled oligonucleotide probes that are complementary to said first and second nucleic acids, respectively, wherein each of said first and second FET labeled oligonucleotides includes a 3′-5′ exonuclease resistant quencher domain; and:
at least one of:
a nucleic acid intercalator, and
a• minor groove binder;
subjecting said primer extension mixture to primer extension reaction conditions;
detecting a change in a fluorescent signal, if any, from said first and second FET labeled oligonucleotide probes to obtain an assay result; and
employing said assay result to determine the presence or absence of said first and second nucleic acids in said sample.
46 . The method according to claim 45 , wherein mixture includes a nucleic acid polymerase having 3′→5′ exonuclease activity.
47 - 53 . (canceled)
54 . The method according to claim 42 , wherein said PCR amplification reaction mixture includes a nucleic acid intercalator and a minor groove binder.
55 . The method according to claim 45 , wherein said primer extension mixture includes a nucleic acid intercalator and a minor groove binder.