Probe based nucleic acid detection
The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that is at least partially complementary to the regulatory sequence, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.
1. 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 fluorescent reporter molecule attached thereto 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 first quencher molecule attached thereto 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 second fluorescent reporter molecule attached thereto 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 second quencher molecule attached thereto and has a melting temperature Tm4,
wherein Tm1, Tm2, Tm3, and Tm4 are higher than an annealing temperature Ta of a PCR amplification reaction.
2. The kit of claim 1 , wherein Ta is in the range of 55-62° C.
3. The kit of claim 1 , wherein Ta is in the range of 60-64° C.
4. The kit of claim 1 , wherein Ta is in the range of 60° C.-62° C.
5. The kit of claim 1 , wherein Ta is 60° C.