Wang M, Lai E
Department of Pharmacology, University of North Carolina at Chapel Hill 27599-7365, USA.
Electrophoresis. 1995 Jan;16(1):1-7. doi: 10.1002/elps.1150160102.
We have studied the separation of large (80-300 kbp) supercoiled (SC) DNA in conventional agarose gel electrophoresis, field inversion gel electrophoresis (FIGE) and pulsed field gel electrophoresis (PFGE). DNA migration was measured under a variety of electrophoretic conditions including different switch times, temperatures, agarose concentrations, and voltage gradients. The migration of SC DNA was found to be inversely proportional to its molecular weight in the three electrophoresis systems tested. In conventional agarose electrophoresis, voltage gradient was found to be the determining parameter in the separation of SC DNA. Unlike large linear DNAs, the migration of SC DNA was found to be independent of switch time in PFGE and FIGE. Broad DNA bands were observed in prolonged FIGE runs. In addition, we have also studied the migration of open-circular (OC) DNA (80 and 100 kbp) in pulsed field gel electrophoresis. Eighty kbp OC DNA can migrate into agarose gels under certain pulsed field conditions whereas 100 kbp OC DNA was trapped at the wells. Based on electrophoretic conditions described in this report, we can determine the size of bacterial artificial chromosome (BAC) clones without restriction enzyme digestion and have enriched the percentage of larger size clones in BAC cloning.
我们研究了在常规琼脂糖凝胶电泳、场反转凝胶电泳（FIGE）和脉冲场凝胶电泳（PFGE）中，大的（80 - 300 kbp）超螺旋（SC）DNA的分离情况。在包括不同切换时间、温度、琼脂糖浓度和电压梯度等多种电泳条件下测量了DNA迁移。在所测试的三种电泳系统中，发现SC DNA的迁移与其分子量成反比。在常规琼脂糖电泳中，发现电压梯度是SC DNA分离的决定性参数。与大的线性DNA不同，在PFGE和FIGE中发现SC DNA的迁移与切换时间无关。在延长的FIGE电泳过程中观察到宽的DNA条带。此外，我们还研究了开环（OC）DNA（80和100 kbp）在脉冲场凝胶电泳中的迁移。在某些脉冲场条件下，80 kbp的OC DNA可以迁移到琼脂糖凝胶中，而100 kbp的OC DNA被困在孔中。基于本报告中描述的电泳条件，我们可以在不进行限制酶消化的情况下确定细菌人工染色体（BAC）克隆的大小，并提高了BAC克隆中较大尺寸克隆的比例。
