Probe based nucleic acid detection

摘要

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.

主权项

1. A method for detecting a target nucleotide sequence comprising:
a) tagging the target nucleotide sequence with a nucleotide tag sequence, thereby producing a tagged target nucleic acid sequence; b) providing a probe oligonucleotide comprising a nucleotide tag recognition sequence complementary to the nucleotide tag sequence and a regulatory sequence 5′ to the nucleotide tag recognition sequence, wherein said probe oligonucleotide comprises a first label and has a melting temperature Tm1; c) amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, wherein said PCR amplification reaction is characterized by an annealing temperature Ta; wherein the PCR amplification reaction is carried out in the presence of a regulatory oligonucleotide comprising a sequence segment that is complementary to the regulatory sequence, wherein said regulatory oligonucleotide comprises a second label and has a melting temperature Tm2; and d) detecting the product of the PCR amplification reaction; wherein the first label and the second label constitute a fluorescent reporter/quencher pair; and wherein Tm1 and Tm2 are both higher than Ta.