Fluorometric Assay Using Dimeric Dyes for Double- and Single-Stranded DNA and RNA with Picogram Sensitivity

✍ Scribed by H.S. Rye; J.M. Dabora; M.A. Quesada; R.A. Mathies; A.N. Glazer

• Book ID: 102965175
• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 535 KB
• Volume: 208
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1020

✦ Synopsis

Thiazole orange homodimer (TOTO; (1,1^{\prime}-(4,4,7,7)-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl2,3-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 \mathrm{ng} / \mathrm{ml}) in the presence of 2 (\times 10^{-7} \mathrm{M}) TOTO or YOYO in (4 \mathrm{mM}) Tris-acetate (/ 0.1 \mathrm{~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 (\mu) l wells in a block of low autofluorescence plastic and detection with a laser-excited confocal fluorescence scanner, as little as (20 \mathrm{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. © 1983 Academic Press. Inc.