NUCLEIC ACID DETECTION USING PROBES

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 complementary to the regulatory sequence and a tail segment that does not hybridize to the probe nucleotide when the sequence segment and the regulatory sequence are annealed, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and using a DNA polymerase with high strand displacement activity and low 5′-nuclease activity, 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 polynucleotide sequence in a sample, comprising:
a) combining
i) a probe oligonucleotide that hybridizes to the target polynucleotide and acts as a forward primer for PCR amplification, comprising a nucleotide tag recognition sequence, a regulatory sequence positioned 5′ to the nucleotide tag recognition sequence, and a reporter moiety;ii) a primer that hybridizes to the target polynucleotide and acts as a reverse primer for PCR amplification;iii) a thermostable DNA polymerase, b) amplifying the target polynucleotide sequence in a PCR amplification reaction using said probe oligonucleotide and said primer, thereby producing an amplicon comprising the regulatory sequence; wherein the PCR amplification reaction is carried out in the presence of a regulatory oligonucleotide comprising
i) a sequence segment that is complementary to the regulatory sequence,ii) a 5′ tail segment that is not complementary to the probe oligonucleotide; andiii) a quencher moiety wherein the reporter moiety and the quencher moiety are fluorescent reporter-quencher pair; wherein the sequence segment of the regulatory oligonucleotide hybridizes to said regulatory sequence in one strand of said amplicon, whereby the signal from the reporter moiety is quenched by quenching moiety; and c) detecting a signal produced by the fluorescent reporter, said signal resulting from the displacement of the regulatory oligonucleotide from the amplicon.