HIGH-SENSITIVITY CAPILLARY ELECTROPHORESIS OF DOUBLE-STRANDED DNA FRAGMENTS USING MONOMERIC AND DIMERIC FLUORESCENT INTERCALATING DYES

Fluorescence-detected capillary electrophoresis separations of phi X17 4/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose so lutions were used as the separation medium. Confocal fluorescence dete ction was performed following 488-nm laser excitation. The limits of D NA detection for on-column staining with monomeric dyes (ethidium brom ide, two propidium dye derivatives, oxazole yellow, thiazole orange, a nd a polycationic thiazole orange derivative) were determined. The thi azole orange dyes provide the most sensitive detection with limiting s ensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from phi X174/HaeIII sam ples containing only 1-2 fg of this fragment per microliter. Separatio ns of preformed DNA-dimeric dye complexes were also performed. The bre adth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numb ers of bound dye molecules that can be resolved as closely spaced subb ands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophor esis with 9-aminoacridine (9AA) in the column and running buffers. The optimum concentrations of 9AA for the separation of complexes preform ed with the dimeric dyes TOTO, EthD, TOTAB, and YOYO were determined t o be 100, 1, 1, and 0.5 mu M, respectively. At these 9AA concentration s, the separations are reproducible, they exhibit excellent resolution , and the 603-bp phi X fragment can be detected following injection fr om DNA solutions as dilute as 40 fg of this DNA fragment per microlite r with TOTO, similar to 100 fg/mu L with YOYO and TOTAB, and 200 fg/mu L with EthD. This work establishes conditions for routine high-sensit ivity fluorescence detection of double-stranded DNA fragments in capil lary electrophoresis. The ability to reliably perform high-quality sep arations of stable noncovalent DNA-dye complexes will also be valuable in the development of multiplex detection methods.