Abstract
The analytical figures of merit of laserโexcited fluorescence detection of DNA restriction fragments, based on the enhanced fluorescence of selected dyes upon intercalation, are presented. The fragments present in a ฯX174 Hae III digest are efficiently separated by capillary electrophoresis employing methyl cellulose as a sizesieving reagent. Onโcolumn DNA labeling is accomplished by adding either ethidium bromide (EB) or POPOโ3 iodide (POPOโ3) to the electrophoretic running buffer. Experimental parameters that are evaluated include type of laser (heliumโcadmium at 325 nm or heliumโneon at 543 nm) and laser power, dye type and concentration, preโinjection labeling procedure, and applied field. Calibration plots are linear over two orders of magnitude in DNA concentration. Limits of detection are in the lowโtoโmid femtogram injected range for the individual fragments, with the POPOโ3 providing slightly lower values. Both dyes exhibit large increases in fluorescence quantum efficiency upon intercalation and contribute little or nothing to the observed optical background. Detectability with the dyes is enhanced by a spectral shift upon intercalation. Signal intensities increase with DNA fragment size in approximately a linear fashion. Intercalation by the dyes also influences mobility and the resolution of the fragments studied.