Fluorometric assay using dimeric dyes for double- and single-stranded DNA and RNA with picogram sensitivity

Thiazole orange homodimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methy l-2,3- dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow homodimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) bind with very high affinity to nucleic acids with more than a 1000-fold fluorescence enhancement upon binding. A linear dependence of fluorescence intensity on DNA concentration over a range from 0.5 to 100 ng/ml in the presence of 2 x 10(-7) M TOTO or YOYO in 4 mM Tris-acetate/0.1 mM EDTA/50 mM NaCl, pH 8.2 allows sensitive quantitation of double-stranded DNA in a conventional fluorometer. With nucleic acid-dye mixtures in an array of 25-microliters wells in a block of low autofluorescence plastic and detection with a laser-excited confocal fluorescence scanner, as little as 20 pg of double-stranded DNA can be detected per well. The array scanning method is rapid, has high throughput, and requires small amounts of sample. It also allows quantitation of single-stranded DNA and RNA.