Universal reporter-based genotyping methods and materials

Abstract

The present disclosure is drawn to methods for detection, quantitation and analysis of nucleotides of interest, for example SNPs, in nucleic acid sequences of interest using universal FRET-based reporter primers.

1 - 14 . (canceled) 
     
     
         15 . A reaction mixture for analyzing, quantitating or detecting one or more alleles or polymorphisms in a target nucleic acid, wherein the reaction mixture comprises a first allele-specific primer, which is specific for a first allele, comprising a first 5′-universal tail which comprises a binding site for a first universal FRET-based reporter primer (UFP), wherein the first UFP comprises a first fluorophore and at least a first quencher moiety; and a second allele-specific primer which is specific for a second allele, comprising a second 5′-universal tail which comprises a binding site for a second UFP, wherein the second UFP comprises a second fluorophore and at least a second quencher moiety; wherein the first fluorophore and the second fluorophore are different and the first quencher moiety and the second quencher moiety are the same or different. 
     
     
         16 . The reaction mixture of  claim 15 , wherein the first fluorophore and the second fluorophore are located at the 5′-end of the first UFP and the second UFP respectively, and the at least first quencher moiety and the at least second quencher moiety are located at an internal nucleotide of the first UFP and the second UFP respectively. 
     
     
         17 . The reaction mixture of  claim 15 , further comprising a locus specific primer. 
     
     
         18 . The reaction mixture of  claim 15 , further comprising one or more nucleic acid polymerases. 
     
     
         19 . The reaction mixture of  claim 15 , further comprising a pyrophosphorolysis enzyme. 
     
     
         20 . The reaction mixture of  claim 15 , further comprising a polyphosphorolysis enzyme. 
     
     
         21 . The reaction mixture of  claim 20 , further comprising one or more polyphosphorolyzing agents 
     
     
         22 - 26 . (canceled) 
     
     
         27 . An oligonucleotide comprising an allele-specific portion and a 5′-universal tail, wherein the 5′-universal tail comprises a binding site for a universal FRET-based reporter primer and the universal tail is not complementary to the target nucleic acid sequence. 
     
     
         28 . The oligonucleotide of  claim 27 , wherein the 5′-universal tail comprises a nucleotide sequence that is identical to the sequence of a universal FRET-based reporter primer. 
     
     
         29 . The oligonucleotide of  claim 27 , wherein the 5′-universal tail comprises a nucleotide sequence that is complementary to the sequence of a universal FRET-based reporter primer. 
     
     
         30 . The oligonucleotide of  claim 27 , wherein the 3′ end of the oligonucleotide comprises a blocking agent such that the blocked end is configured to be activated by a pyrophosphorolysis enzyme. 
     
     
         31 . The oligonucleotide of  claim 30  wherein the blocking agent is a dideoxynucleotide (ddN). 
     
     
         32 . A kit for analyzing, quantitating or detecting one or more alleles or polymorphisms in a target nucleic acid, wherein the kit comprises a first allele-specific primer, which is specific for a first allele, comprising a first 5′-universal tail which comprises a binding site for a first universal FRET-based reporter primer (UFP), wherein the first UFP comprises a first fluorophore and at least a first quencher moiety; and a second allele-specific primer which is specific for a second allele, comprising a second 5′-universal tail which comprises a binding site for a second UFP, wherein the second UFP comprises a second fluorophore and at least a second quencher moiety; wherein the first and second fluorophore are different and the at least first quencher moiety and the at least second quencher moiety are the same or different. 
     
     
         33 . The kit of  claim 32 , wherein the first fluorophore and the second fluorophore are located at the 5′-end of the first UFP and the second UFP respectively, and the at least first quencher moiety and the at least second quencher moiety are located at an internal nucleotide of the first UFP and the second UFP respectively. 
     
     
         34 . The kit of  claim 32 , further comprising a locus specific primer. 
     
     
         35 . The kit of  claim 32 , further comprising one or more nucleic acid polymerases. 
     
     
         36 . The kit of  claim 32 , further comprising a pyrophosphorolysis enzyme. 
     
     
         37 . The kit of  claim 32 , further comprising a pyrophosphorolysis enzyme. 
     
     
         38 . The kit of  claim 37 , further comprising one or more polyphosphorolyzing agents.