A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA

DNA separations were performed in poly(ethylene oxide) (PEO) solutions prep ared in 100 mM Tris-boric acid (TB) buffers using a capillary filled with T B buffers with concentrations up to 2.5 M, pH 10.0. The electroosmotic flow (EOF) increased with increasing the concentration of TB buffers till 1.5 M as a result of decreasing PEO adsorption on the capillary wall. At high TB concentrations (> 1.5 M), the peaks corresponding to small DNA fragments ( 11 and 8 base pairs) became sharper and were detected. Relative standard de viations of the EOF coefficient and the migration times of the DNA fragment s were all less than 1% using a capillary filled with TB buffers at concent rations higher than 1.5 M. When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitiv ity, speed, and resolution were generally achieved. The fluorescence intens ity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutio ns 10.0/8.2 was 27-fold of that at pH values 8.2/8.2. The enhancement was r elated to effects of pH and berate on fluorescence intensity, DNA conformat ion, stacking, and interactions with the capillary wall. Using a capillary filled with 400 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeI II digest, PER 328/Bgl1 digest and pBR 328/Hinfl digest) in 1.5% PEO soluti ons prepared in 100 mu TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.