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

Abstract

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 4x174 Hae I11 digest are efficiently separated by capillary electrophoresis employing methyl cellulose as a sizesieving reagent. On-column DNA labeling is accomplished by adding either ethdium 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.

Highly reproducible capillary gel electrophoresis (CGE) of DNA fragments using uncoated columns. Detection of genetically modified maize by PCR-cGE

“…As can be seen, slightly longer analysis times were obtained for the Bt-DNA and MSS-DNA fragments when CGE-LIF was used (Figure 3.A) compared to the migration times with CGE-UV ( Figure 2). This effect has been already observed and attributed to the effect of the intercalating dye on the electrophoretic mobility of DNA fragments [27]. In good agreement, this increase in migration times was also observed for the DNA ladder injected under the same conditions as demonstrated in Figure 3.B where, moreover, all the DNA fragments are adequately separated.…”

Ultra‐fast simultaneous analysis of genetically modified organisms in maize by microchip electrophoresis with LIF detector

“…Fluorescence detection has high sensitivity, intrinsic simplicity, and can allow miniaturization of the separation platforms onto planar chips for sequence detection of DNA due to the smaller sample injection volume. The concentration of the dye, EtBr, is important for the fluorescence detection of the dye-DNA complexes because it significantly increases the detection sensitivity without influencing the migration pattern of the DNA fragments [30,31]. Therefore, the effect of the EtBr dye concentration on the detection sensitivity and migration of the DNA fragments was also investigated at concentrations ranging from 0.1 to 1.5 mg/mL.…”

Optimization of separation conditions in the size-selective capillary electrophoretic separation of DNA fragments

“…Capillary efficiency is expressed as total plate count ( N ) and given by the effective length of the capillary ( 1 ) divided by the observed plated height (H). While we have found that capillary efficiencies can vary from day to day, typical plate heights are 0.3-0.4 p m and do not seem to depend on [MC] or other adjustable parameters.…”