Tseng W L, Chang H T
Department of Chemistry, National Taiwan University, Taipei.
Electrophoresis. 2001;22(4):763-70. doi: 10.1002/1522-2683(200102)22:4<763::AID-ELPS763>3.0.CO;2-W.
DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 mM 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 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/I digest and pBR 328/HinfI digest) in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.
在100 mM Tris-硼酸（TB）缓冲液中制备的聚环氧乙烷（PEO）溶液中进行DNA分离，使用填充有浓度高达2.5 M、pH 10.0的TB缓冲液的毛细管。由于PEO在毛细管壁上的吸附减少，电渗流（EOF）随着TB缓冲液浓度的增加而增加，直至1.5 M。在高TB浓度（>1.5 M）下，对应于小DNA片段（11和8个碱基对）的峰变得更尖锐并被检测到。使用填充有浓度高于1.5 M的TB缓冲液的毛细管时，EOF系数和DNA片段迁移时间的相对标准偏差均小于1%。当在PEO溶液和TB缓冲液的不同pH值下进行分离时，通常在灵敏度、速度和分辨率方面能获得更好的结果。在TB缓冲液/PEO溶液pH值为10.0/8.2时获得的2176 bp片段的荧光强度是pH值为8.2/8.2时的27倍。这种增强与pH和硼酸盐对荧光强度、DNA构象、堆积以及与毛细管壁相互作用的影响有关。使用填充有400 mM TB缓冲液（pH 10.0）的毛细管，在375 V/cm下，在100 mM TB缓冲液（pH 9.0）中制备的1.5% PEO溶液中对DNA（pBR 322/HaeIII酶切产物、pBR 328/Bg/I酶切产物和pBR 328/HinfI酶切产物）的分离在不到18分钟内完成。
