Liang C, Gerbi S A
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
Mol Cell Biol. 1994 Feb;14(2):1520-9. doi: 10.1128/mcb.14.2.1520-1529.1994.
The replication origin region for DNA amplification in Sciara coprophila DNA puff II/9A was analyzed with a novel three-dimensional (3D) gel method. Our 3D gel method involves running a neutral/neutral 2D gel and then cutting out vertical gel slices from the area containing replication intermediates, rotating these slices 90 degrees to form the third dimension, and running an alkaline gel for each of the slices. Therefore, replication intermediates are separated into forks and bubbles and then are resolved into parental and nascent strands. We used this technique to determine the size of forks and bubbles and to confirm the location of the major initiation region previously mapped by 2D gels to a 1-kb region. Furthermore, our 3D gel analyses suggest that only one initiation event in the origin region occurs on a single DNA molecule and that the fork arc in the composite fork-plus-bubble pattern in neutral/neutral 2D gels does not result from broken bubbles.
利用一种新型的三维（3D）凝胶方法，对嗜粪Sciara DNA膨泡II/9A中用于DNA扩增的复制起始区域进行了分析。我们的3D凝胶方法包括进行中性/中性二维凝胶电泳，然后从包含复制中间体的区域切出垂直凝胶条带，将这些条带旋转90度形成第三维，并对每个条带进行碱性凝胶电泳。因此，复制中间体被分离为叉状结构和泡状结构，然后被解析为亲本链和新生链。我们使用该技术确定叉状结构和泡状结构的大小，并确认先前通过二维凝胶电泳定位到1kb区域的主要起始区域的位置。此外，我们的3D凝胶分析表明，在起始区域单个DNA分子上仅发生一次起始事件，并且中性/中性二维凝胶中复合叉状加泡状模式中的叉状弧不是由破裂的泡状结构产生的。
