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

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.

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.