This detection method relies on FRET of two adjacent oligonucleotide probes (Fig. 18.2b). When both probes are specifically bound to the target amplicon, the energy emitted by the donor dyes excites the acceptor dye of the second probe, which then emits fluorescent light at a longer wavelength. One probe is labeled with a donor fluorochrome (fluorescein) at the 3' end, and the other probe is labeled with an acceptor dye (Cy5, LC Red 640) at the 5' end. Both probes can hybridize to the target sequences, and the two probes are usually no more than 3 bases apart (4 to 25 A molecular distance). The first dye (fluorescein) is excited by the LED (light emitting diode) filtered light source and emits green fluorescent light at a slightly longer wavelength. When the two dyes are in close proximity as the probes simultaneously hybridize to their target, the emitted energy excites the acceptor (i.e., LC Red 640) attached to the second hybridization probe that subsequently emits red fluorescent light at a longer wavelength. The occurrence of FRET is characterized by a decrease in observed donor emission and a simultaneously increased acceptor emission. The ratio between donor fluorescence and acceptor fluorescence increases during the PCR and is proportional to the amount of target DNA generated (Wittwer, 1997; Nitsche, 1999).
An advantage hybridization probes thus have over the hydrolysis probes is their relative tolerance to singlebase variations; therefore their suitability for genotyping in combination with melt-peak analysis. A disadvantage is the need for a larger sequence area necessary to accommodate two adjacent probes.
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