Huang M F, Hsu C E, Tseng W L, Lin Y C, Chang H T
Department of Chemistry, National Taiwan University, Taipei, ROC.
Electrophoresis. 2001 Jul;22(11):2281-90. doi: 10.1002/1522-2683(20017)22:11<2281::AID-ELPS2281>3.0.CO;2-C.
Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.
使用包括线性聚丙烯酰胺（LPA）、聚环氧乙烷（PEO）、羟丙基纤维素（HPC）、羟乙基纤维素（HEC）和琼脂糖在内的五种不同聚合物溶液，在电渗流（EOF）存在的情况下对φX-174/HaeIII DNA限制片段进行了分离。在分离过程中，聚合物溶液通过电渗流进入毛细管。使用LPA溶液时，由于在毛细管壁上的吸附，整体电渗流较小。另一方面，使用衍生纤维素和PEO溶液对大的DNA片段（>872个碱基对，bp）的分离更快且更好。已经开发了几种通过控制电渗流和/或改变DNA的电泳迁移率来实现最佳分辨率和速度的方法，包括（i）溴化乙锭（0.5 - 5μg/mL）的逐步变化，（ii）电压编程（125 - 375 V/cm），（iii）使用混合聚合物溶液，以及（iv）使用高浓度的Tris - 硼酸盐（TB）缓冲液。在等度条件下使用2% PEO（8,000,000）未分离的434至653 bp的DNA片段，通过溴化乙锭的逐步变化或电压编程已完全分离。与PEO溶液相比，由PEO和HEC制备的混合聚合物溶液具有更高的分辨能力。使用填充有600 mM TB缓冲液（pH 10.0）的毛细管，在1.5% PEO中实现了DNA（pBR 322/HaeIII酶切、pBR 328/Bg/l酶切和pBR 328/Hinfl酶切）的高速（<15分钟）分离。
