Steve Hilliard wrote: >I have a user who's working out a DNA binding competition assay, and >they're looking at detection methods. They were thinking about incubating >oligo-labelled beads with a fluorescent complimentary strand, >and then incubating with the analyte. Competition would knock off the >label and reduce fluorescence. Now they're thinking about starting with >beads with fluorescently labelled oligos, incubating them with >complimentary probes conjugated to quenching molecules (blocking the >bead fluorescence), and then doing the analyte incubation. Successful >competition would then knock off the quenchers and increase fluorescence. > >Any opinions ? I'm aware that Trypan blue quenches FITC, but is >this a binary (on/off) phenomenon? Does the degree of quenching >vary? Can trypan blue be stably conjugated to oligos? Are there other >molecules out there to consider, or any source of info that someone could >recomend? My gut impression is that it won't make much of a >difference--it seems to me that our signal/noise ratio is going to >depend on how many FITC molecules are knocked off (protocol a) or >exposed (protocol b). I can't see why one approach would be better >than the other, and if the quenching response varies at all it seems like >it just introduces one more source of variation. That's right. Quenching is almost certainly harder to control than fluorescence- if there is a well-developed system out there, then it may be usable, but, if your user is going to have to develop one, he or she would be better off with the straight competitive binding assay between the nonfluorescent analyte and the fluorescent complementary strand. -Howard
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