DETECTION OF NUCLEIC ACIDS BY FLUORESCENCE QUENCHING

摘要

A detector oligonucleotide having a sequence which forms an intramolecularly base-paired secondary structure is described for use in detecting nucleic acid t arget sequences and target sequence amplification. The detector oligonucleotide is further modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned on th e detector oligonucleotide such that they are in close spatial proximity in the base-paired , folded secondary structure, thereby causing quenching of donor fluorescence. The detect or oligonucleotide may optionally further comprise a restriction endonuclease recog nition site (RERS) which remains partially or entirely single-stranded in the base-paired se condary structure. The RERS is flanked by the two dyes. In the presence of target, the b ase-paired secondary structure is unfolded or linearized, increasing the distance between t he donor and acceptor dyes and causing a change in fluorescence of the donor and/or the accep tor. If an RERS is present, it is rendered double-stranded in the presence of target, allow ing cleavage or nicking by a restriction endonuclease and separation of the two dyes onto separa te nucleic acid fragments. This may further contribute to the magnitude of the change in fluores cence.