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A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA
Author(s) -
Tseng WeiLung,
Chang HuanTsung
Publication year - 2001
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200102)22:4<763::aid-elps763>3.0.co;2-w
Subject(s) - capillary electrophoresis , capillary action , chemistry , chromatography , fluorescence , analytical chemistry (journal) , boric acid , stacking , ethylene oxide , adsorption , dna , electrophoresis , materials science , polymer , copolymer , biochemistry , physics , organic chemistry , quantum mechanics , composite material
DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 m M Tris‐boric acid (TB) buffers using a capillary filled with TB 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 deviations of the EOF coefficient and the migration times of the DNA fragments were all less than 1% using a capillary filled with TB buffers at concentrations higher than 1.5 M . When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitivity, speed, and resolution were generally achieved. The fluorescence intensity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutions 10.0/8.2 was 27‐fold of that at pH values 8.2/8.2. The enhancement was related to effects of pH and borate on fluorescence intensity, DNA conformation, stacking, and interactions with the capillary wall. Using a capillary filled with 400 m M TB buffers, pH 10.0, the separation of DNA (pBR 322/ Hae III digest, pBR 328/ Bgl I digest and pBR 328/ Hin fI digest) in 1.5% PEO solutions prepared in 100 m M TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.