Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning.
Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning.
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大型超螺旋和开环 DNA 的脉冲场分离及其在细菌人工染色体克隆中的应用。
DOI：
10.1002/elps.1150160102
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Lai,E
中科院分区：
文献类型：
--
作者：
Wang,M;Lai,E
我们研究了传统琼脂糖凝胶电泳、反转场凝胶电泳 (FIGE) 和脉冲场凝胶电泳 (PFGE) 中大 (80–300 kbp) 超螺旋 (SC) DNA 的分离。 DNA 迁移在多种电泳条件下进行测量，包括不同的转换时间、温度、琼脂糖浓度和电压梯度。在所测试的三个电泳系统中，发现 SC DNA 的迁移与其分子量成反比。在传统的琼脂糖电泳中，电压梯度被发现是 SC DNA 分离的决定参数。与大的线性 DNA 不同，SC DNA 的迁移被发现与 PFGE 和 Fige 中的转换时间无关。在长时间的 Fige 运行中观察到宽的 DNA 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 OC DNA 可以迁移到琼脂糖凝胶中，而 100 kbp OC DNA 则被捕获在孔中。根据本报告中描述的电泳条件，我们可以在不进行限制性酶消化的情况下确定细菌人工染色体（BAC）克隆的大小，并丰富了 BAC 克隆中较大尺寸克隆的百分比。
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.