Fluorescent DNA Probes Fluorescence-based methods for detecting nucleic acids are an attractive alternative to traditional radioactive and colorimetric methods. Single-labeled fluorescent oligonucleotides are routinely used in such applications as DNA sequencing, genotyping, sequence detection and enzyme assays. Dual-labeled oligonucleotide designs, where a fluorescent reporter and a fluorescence quencher are placed on the same oligonucleotide, enable closed-tube systems which permit rapid and sensitive high-throughput assays. Most dual-labeled fluorescent systems employ FRET principles to control the dark versus bright state. Fluorescence Resonance Energy Transfer (FRET) is a process where energy from an excited fluorophore is transferred to a neighboring acceptor molecule without the release of light by the fluorophore (quenching). In FRET, the efficiency of this energy transfer process is dependent upon the physical distance between the reporter fluorophore and the quencher and the degree of overlap between reporter emission and quencher absorption spectra. When reporter and quencher are close, quenching is efficient and the oligo is dark. When reporter and quencher are distant, quenching is reduced or eliminated and the oligo is bright. The range over which quenching occurs is unique for each reporter/quencher pair but is commonly between 30 and 70 Å (the distance where quenching is 50% efficient is defined as the Förster radius for that R/Q pair). The quencher (energy acceptor) molecule can be another fluorescent dye such as TAMRATM or a non-fluorescent dark quencher. 
Probes incorporating dark quenchers have lower background fluorescence, providing greater sensitivity. Since dark quenchers absorb broadly and do not emit light, multiple reporter dyes can be used with a given quencher, expanding the options available for multiplex assays. Dark quenchers simplify detection, making them compatible with a broad range of image analysis instruments.
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