Nucleic Acids Research, 1992, Vol. 20, No. 11 2803-2812
© 1992
CHEMISTRY |
Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications
1Department of Molecular and Cell Biology, University of California Berkeley, CA 94720, 2Molecular Probes, Inc., Eugene, OR 97402 3Department of Chemistry, University of California Berkeley,CA 94720 4Division of Chemical Biodynamics, Lawrence Berkeley Laboratory Berkeley, CA 94720, USA
*To whom correspondence should be addressed at MCB:Stanley/Donner ASU, 229 Stanley Hall, University of California, Berkeley, CA 94720, USA
Received February 12, 1992. Accepted May 1, 1992.
The 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-methyl-idenel-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 (
max 513 nm;
532 nm) and dsDNA-YOYO (max 489 nm; 509 nm) complexes are completely stable to electrophoresis agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; 616 nm) and those prelabeled 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, quantitation, 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 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.
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