FLUORESCENCE ENERGY-TRANSFER CYANINE HETERODIMERS WITH HIGH-AFFINITY FOR DOUBLE-STRANDED DNA .2. APPLICATIONS TO MULTIPLEX RESTRICTION FRAGMENT SIZING

Energy-transfer cyanine dyes, a thiazole orange-thiazole-indolenine (b utyl TOTIN) and a thiazole orange-thiazole blue heterodimer (pentyl TO TAB), form high-affinity complexes with double-stranded (ds) DNA with donor fluorescence (lambda(max)(F) 527 nm) quenched >90% and with acce ptor fluorescence emission above 650 nm (S. C. Benson, Z. Zeng, and A. N. Glazer (1995) Anal. Biochem. 231, 247-255). After separation by ag arose gel electrophoresis, bands of precomplexed dsDNA-dye restriction fragments containing 10 pg of dsDNA are readily detected with a laser -excited confocal-fluorescence gel scanner (R. A. Mathies et al. (1994 ) Rev. Sci. Instrum. 65, 807-812) following donor excitation at 488 nm (argon ion laser) or direct acceptor excitation at 647 nm (krypton io n laser). Accurate two-color multiplex sizing of restriction fragments is obtained with 488-nm excitation with dsDNA fragments precomplexed with thiazole orange dimer (TOTO) as unknowns (detected at 500-565 nm, green channel) and dsDNA fragments stained with the energy-transfer c yanine dyes (detected at 645-750 nm, red channel) as internal standard s, There is negligible cross talk of fluorescence between the red and green channels and no significant dye migration in mixtures of prelabe led standard and unknown fragments. (C) 1995 Academic Press, Inc.