Evaluation of a sheath flow cuvette for postcolumn fluorescence derivatization of DNA fragments separated by capillary electrophoresis.

The investigation and evaluation of the sheath flow cell as a reaction chamber to postcolumn fluorescently derivatize DNA fragments separated by capillary electrophoresis is described herein. Use of the sheath flow cell arrangement facilitates the mixing of the intercalating dye, ethidium bromide (EB), and the effluent from the separation capillary by diffusion without a high degree of band dispersion. Theoretical plate counts of >1 × 10(6) are reported with the postcolumn derivatization technique, and resolution of all of the fragments in a φx-174-HaeIII digest is achieved. Optimization of experimental parameters such as flow rate, position of the detection zone, and EB concentration is examined. A limit of detection in the low nanograms-per-milliliter range with a linear dynamic range over 3 orders of magnitude is reported for a sample of φx-174-HaeIII digest. Evaluation of postcolumn derivatization for the investigation of DNA-protein interactions is demonstrated. The integrity of a DNA-trp-repressor protein interaction is maintained with the postcolumn approach but is compromised when EB is added to the running buffer.