Evaluation of on-column labeling with intercalating dyes for fluorescence detection of DNA fragments separated by capillary electrophoresis

Abstract

The analytical figures of merit of laser-excited fluorescence detection of DNA restriction fragments, based on the enhanced fluorescence of selected dyes upon intercalation, are presented. The fragments present in a ϕX174 Hae III digest are efficiently separated by capillary electrophoresis employing methyl cellulose as a sizesieving reagent. On-column DNA labeling is accomplished by adding either ethidium bromide (EB) or POPO-3 iodide (POPO-3) to the electrophoretic running buffer. Experimental parameters that are evaluated include type of laser (helium-cadmium at 325 nm or helium-neon at 543 nm) and laser power, dye type and concentration, pre-injection labeling procedure, and applied field. Calibration plots are linear over two orders of magnitude in DNA concentration. Limits of detection are in the low-to-mid femtogram injected range for the individual fragments, with the POPO-3 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.