Primers and methods for the detection and discrimination of nucleic acids

Abstract

The present invention provides novel primers and methods for the detection of specific nucleic acid sequences. The primers and methods of the invention are useful in a wide variety of molecular biology applications and are particularly useful in allele specific PCR.

1 .- 74 . (canceled) 
     
     
         75 . A composition comprising at least one nucleic acid molecule and at least two oligonucleotides, wherein at least a portion of said first and second oligonucleotides are capable of hybridizing with at least a portion of said nucleic acid molecule and wherein said first and second oligonucleotides each comprise at least one modified nucleotide internally and/or at or near the 3′- and/or 5′-terminal nucleotide. 
     
     
         76 . The composition of  claim 75 , wherein said at least one modified nucleotide of said first and/or said second oligonucleotide is a 2′-O-alkyl ribonucleotide. 
     
     
         77 . The composition of  claim 75 , said composition further comprising at least one component selected from the group consisting of one or more nucleotides, one or more DNA polymerases and one or more reverse transcriptases. 
     
     
         78 . The composition of  claim 75 , wherein said modified nucleotide of said first oligonucleotide comprises a detectable label. 
     
     
         79 . The composition of  claim 78 , wherein said detectable label is a fluorescent moiety. 
     
     
         80 . The composition of  claim 79 , wherein said modified nucleotide on said second oligonucleotide is a quencher moiety. 
     
     
         81 . The composition of  claim 80 , wherein said fluorescent moiety and/or said quencher moiety is located at a 3′-terminal nucleotide on said first and/or said second oligonucleotide. 
     
     
         82 . The composition of  claim 80 , wherein said fluorescent moiety and/or said quencher moiety is located at a 5′-terminal nucleotide on said first and/or said second oligonucleotide. 
     
     
         83 . The composition of  claim 75 , wherein said nucleic acid molecule comprises a RNA molecule. 
     
     
         84 . The composition of  claim 75 , wherein said nucleic acid molecule comprises a cDNA molecule. 
     
     
         85 . The composition of  claim 80 , wherein said fluorescent moiety and said quencher moiety comprise a FRET pair. 
     
     
         86 . The composition of  claim 79 , wherein said fluorescent moiety is selected from the group consisting of a nuoresceinphosphoramidite (FAM) moiety, a 6-carboxynuorescein succinimidyl ester (6-FAM, SE) moiety, a nuorescein-5-isothiocyanate (FITC) moiety, a 5-(6-)-carboxytetramethyl rhodamine (TAMRA) moiety, a succinimidyl ester moiety, and a 4,4-dinuoro-5, 7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY 530/550) moiety. 
     
     
         87 . A method for quantitation or detection of one or more target nucleic acid molecules in a nucleic acid sample during nucleic acid amplification comprising:
 mixing said nucleic acid sample with a composition comprising at least two oligonucleotides under conditions sufficient to amplify said one or more target nucleic acid molecules complementary to, all or a portion of a sequence of said target nucleic acid molecule(s), said amplified target nucleic acid molecule(s) comprising said oligonucleotides, wherein said first oligonucleotide comprises a detectable label located internally and/or at or near a 3′- and/or 5′-terminal nucleotide; and   detecting the presence or absence or quantifying the amount of said target nucleic acid molecule(s) by measuring said detectable label of said first oligonucleotide.   
     
     
         88 . The method of  claim 86 , wherein said second oligonucleotide comprises a quencher moiety located internally and/or at or near a 3′- and/or 5′-terminal nucleotide 
     
     
         89 . A kit for use in the synthesis and/or amplification of a target nucleic acid molecule, said kit comprising at least two oligonucleotides, wherein at least a portion of said first and second oligonucleotides are capable of hybridizing with at least a portion of said target nucleic acid molecule and wherein said first and second oligonucleotides each comprise at least one modified nucleotide internally and/or at or near the 3′- and/or 5′-terminal nucleotide. 
     
     
         90 . The kit of  claim 89 , wherein said at least two oligonucleotides are provided in a single composition or mixture of said kit. 
     
     
         91 . The kit of  claim 89 , wherein said modified nucleotide of said first oligonucleotide comprises a detectable label. 
     
     
         92 . The kit of  claim 91 , wherein said detectable label is a fluorescent moiety. 
     
     
         93 . The composition of  claim 92 , wherein said modified nucleotide on said second oligonucleotide is a quencher moiety. 
     
     
         94 . The kit of  claim 93 , wherein said fluorescent moiety and said quencher moiety comprise a FRET pair.