Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.
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-thiaz ole)-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 greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 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, 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.
Rye
H S
HS
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Yue
S
S
Wemmer
D E
DE
Quesada
M A
MA
Haugland
R P
RP
Mathies
R A
RA
Glazer
A N
AN
D001583:Benzoxazoles / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D004996:Ethidium / Q000737:chemistry; D005456:Fluorescent Dyes; D007364:Intercalating Agents / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010080:Oxazoles / Q000737:chemistry*; D011806:Quinolinium Compounds / Q000737:chemistry*; D015183:Restriction Mapping; D013844:Thiazoles / Q000737:chemistry*
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D010080:Oxazoles; D011806:Quinolinium Compounds; D013844:Thiazoles; C075295:1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene)quinolinium; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; D004996:Ethidium