DETECTION OF NUCLEIC ACIDS BY FLUORESCENCE QUENCHING

摘要

Single-stranded signal primers are modified by linkage to two dyes which for m a donor/acceptor dye pair. The two dyes are positioned in sufficiently close s patial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition si te (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not clea vable or nickable by the restriction endonuclease. In the presence of target, however, signal pri mer and the restriction endonuclease recognition site are rendered double-stranded and c leavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indicati on of the presence of the target sequence or of target sequence amplification.