Department of Biology, Tufts University;
Department of Biology, Tufts University.
J Vis Exp. 2024 Sep 13(211). doi: 10.3791/67229.
Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.
二维中性/中性凝胶电泳（2DGE）已成为分析通过自然障碍进行 DNA 复制的基准技术。本方案描述了如何通过人类细胞中基于猿猴病毒 40（SV40）的附加体中的结构倾向、可扩展 DNA 重复来分析复制叉的进展。简而言之，在将质粒转染到人类细胞后，通过改良的 Hirt 方案分离复制中间体，并使用 DpnI 限制酶处理以去除未复制的 DNA。然后用适当的限制酶消化中间体，将感兴趣的重复序列置于 3-5 kb 长 DNA 片段的原点-远端半部分内。复制中间体首先按大小，然后按形状在两个垂直维度上分离。Southern 印迹杂交后，该方法允许研究人员观察在复制 Y 形臂下降半部分的各种结构形成重复处的叉停顿。此外，这种停顿位置可以可视化重复介导的叉停顿的各种结果，例如叉反转、汇聚叉的出现和重组叉的重新启动。
