Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications
1992; Oxford University Press; Volume: 20; Issue: 11 Linguagem: Inglês
10.1093/nar/20.11.2803
ISSN1362-4962
AutoresHays S. Rye, Stephen Yue, David E. Wemmer, Mark A. Quesada, Richard P. Haugland, Richard A. Mathies, Alexander N. Glazer,
Tópico(s)Metal complexes synthesis and properties
ResumoThe synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1 '- (4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1 ,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3- oxazole in place of the benzo-1,3-thiazole) are reported.TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with >1000-fold fluorescence enhancement.The dsDNA-TOTO (Xmax 513 nm; miax 532 nm) and dsDNA-YOYO (Xmax 489 nm; Xax 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels.Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; max 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis.Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620 -750 nm (dsDNA-EthD emission) and 500 -565 nm (dsDNA-TOTO or dsDNA- YOYO emission) allowed sensitive detection, quantita- tion, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp.The limit of detection of dsDNA-TOTO and YOYO complexes with a laser- excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 pico- grams, about 500-fold lower than attainable by con- ventional staining with ethidium bromide.
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