Staib C, Grummt F
Institute of Biochemistry, University of Würzburg, Germany.
Methods. 1997 Nov;13(3):293-300. doi: 10.1006/meth.1997.0528.
The mapping of replication origins by nascent DNA strand length determination is a very sensitive generally applicable method that identifies even single-copy origins in mammalian chromosomes. A major advantage of this procedure is that there is no need for synchronization of cells or treatment with metabolic agents, which allows the origin to be studied under physiological conditions. This technique is based upon the amplification of specific sequence markers on nascent DNA strands that initiated replication within the region of the putative origin. Therefore, this method requires detailed sequence information of the locus to be analyzed. As a first step, nascent DNA of proliferating cells is pulse-labeled with BrdU followed by size fractionation and purification with anti-BrdU antibodies. The position of putative origins can then be determined via identification of the shortest nascent strands that can be amplified by PCR and hybridized to probes homologous to the amplified segments. Here, we give a detailed description of the theory behind the method and a full recipe for its application. Advantages and limitations of the procedure are discussed.
通过新生DNA链长度测定来绘制复制起点是一种非常灵敏且普遍适用的方法，它甚至能够识别哺乳动物染色体中的单拷贝起点。该方法的一个主要优点是无需对细胞进行同步化处理或用代谢剂进行处理，这使得能够在生理条件下研究起点。此技术基于对新生DNA链上特定序列标记的扩增，这些标记在假定起点区域内启动复制。因此，该方法需要待分析位点的详细序列信息。第一步，用溴脱氧尿苷（BrdU）对增殖细胞的新生DNA进行脉冲标记，随后进行大小分级分离并用抗BrdU抗体进行纯化。然后，通过鉴定能够通过聚合酶链反应（PCR）扩增并与与扩增片段同源的探针杂交的最短新生链，来确定假定起点的位置。在此，我们详细描述该方法背后的理论及其应用的完整方案。同时还讨论了该方法的优缺点。
