Probe based nucleic acid detection

A kit comprising:

• a) a first tagging primer comprising a first nucleotide tag sequence and a first target nucleotide recognition sequence that hybridizes to a first allele at a polymorphic site in a target nucleic acid sequence;
• b) a second tagging primer comprising a second nucleotide tag sequence and a second target nucleotide recognition sequence that hybridizes to a second allele at the polymorphic site in the target nucleic acid sequence;
• c) a reverse primer;
• d) a first probe oligonucleotide comprising (i) a first nucleotide tag recognition sequence complementary to the first nucleotide tag sequence and (ii) a first regulatory sequence 5′ to the first nucleotide tag recognition sequence, wherein the first probe oligonucleotide comprises a first label and has a melting temperature Tm1;
• e) a first regulatory oligonucleotide comprising a first sequence segment that is complementary to the first regulatory sequence, wherein the first regulatory oligonucleotide comprises a second label and has a melting temperature Tm2;
• f) a second probe oligonucleotide comprising (i) a second nucleotide tag recognition sequence complementary to the second nucleotide tag sequence and (ii) a second regulatory sequence 5′ to the second nucleotide tag recognition sequence, wherein the second probe oligonucleotide comprises a third label and has a melting temperature Tm3;
• and
• g) a second regulatory oligonucleotide comprising a second sequence segment that is complementary to the second regulatory sequence, wherein the second regulatory oligonucleotide comprises a fourth label and has a melting temperature Tm4,
• wherein Tm1, Tm2, Tm3, and Tm4 are higher than an annealing temperature Ta of a PCR amplification reaction.

A PCR amplification reaction comprising:

• a) a first tagged target nucleic acid sequence comprising a first allele at a polymorphic site and a first nucleotide tag sequence;
• b) a first probe oligonucleotide comprising (i) a first nucleotide tag recognition sequence complementary to the first nucleotide tag sequence and (ii) a first regulatory sequence 5′ to the first nucleotide tag recognition sequence, wherein the first probe oligonucleotide comprises a first label and has a melting temperature Tm1;
• c) a first regulatory oligonucleotide comprising a first sequence segment that is complementary to the first regulatory sequence, wherein the first regulatory oligonucleotide comprises a second label and has a melting temperature Tm2;
• d) a second tagged target nucleic acid sequence comprising a second allele at the polymorphic site and a second nucleotide tag sequence;
• e) a second probe oligonucleotide comprising (i) a second nucleotide tag recognition sequence complementary to the second nucleotide tag sequence and (ii) a second regulatory sequence 5′ to the second nucleotide tag recognition sequence, wherein the second probe oligonucleotide comprises a third label and has a melting temperature Tm3;
• and
• f) a second regulatory oligonucleotide comprising a second sequence segment that is complementary to the second regulatory sequence, wherein the second regulatory oligonucleotide comprises a fourth label and has a melting temperature Tm4,
• wherein the PCR amplification reaction is characterized by an annealing temperature Ta, wherein the first label and the second label constitute a first fluorescent reporter/quencher pair, wherein the third label and the fourth label constitute a second fluorescent reporter/quencher pair, and wherein Tm1, Tm2, Tm3, and Tm4 are higher than Ta.