Anal. Chem. 1998, 70, 182-186
Technical Notes
Evaluation of a Sheath Flow Cuvette for Postcolumn Fluorescence Derivatization of DNA Fragments Separated by Capillary Electrophoresis William F. Nirode,† Tracy D. Staller,† Roderic O. Cole,*,‡ and Michael J. Sepaniak*,†
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, and Pfizer Central Research, Drug Discovery, Eastern Point Road, Groton, Connecticut 06340
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 × 106 are reported with the postcolumn derivatization technique, and resolution of all of the fragments in a Ox-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 Ox-174-HaeIII digest. Evaluation of postcolumn derivatization for the investigation of DNA-protein interactions is demonstrated. The integrity of a DNA-trprepressor protein interaction is maintained with the postcolumn approach but is compromised when EB is added to the running buffer. Capillary electrophoresis (CE) has become an important analytical technique with many applications, ranging from inorganic ions1,2 to large biomolecules such as DNA and proteins.3-8 CE, characterized by its high efficiencies, speed, and small sample quantities, is well suited for the study of DNA-protein interactions. The analysis of DNA-protein interactions between the trp repressor of Escherichia coli (protein) and the trp operator (DNA) †
University of Tennessee. Pfizer Central Research. (1) Jones, W. R.; Jandik, P. J. Chromatogr. 1991, 546, 445-458. (2) Weston, A.; Brown, P. R.; Jandik, P.; Jones, W. R.; Heckenberg, A. L. J. Chromatogr. 1992, 593, 289-295. (3) Fung, E. N.; Yeung, E. S. Anal. Chem. 1996, 67, 1913-1919. (4) Strege, M. A.; Lagu, A. L. J. Chromatogr. 1993, 630, 337-344. (5) Novotony, M. V.; Cobb, K. A.; Liu, J. Electrophoresis 1990, 11, 735-742. (6) Swaile, D. F.; Sepaniak, M. J. J. Liquid Chromatogr. 1991, 14 (5), 869893. (7) Chen, F.-T. A. J. Chromatogr. 1991, 559, 445-453. (8) De Antonis, K. M.; Brown, P. R.; Chemg, Y.-F.; Cohen, S. A. J. Chromatogr. A 1994, 661, 279-285. ‡
182 Analytical Chemistry, Vol. 70, No. 1, January 1, 1998
has been demonstrated.9 The DNA binding in the trp repressor/ trp operator system is regulated by allosteric control; thus, derivatization of the DNA with an intercalating dye will significantly affect the DNA-protein interaction. Detection in CE is usually performed on-column, where the detection zone is limited to the cross section over a short segment (
