Genome-scale identification of active DNA replication origins
Genome-scale identification of active DNA replication origins
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DOI：
10.1016/j.ymeth.2012.06.015
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发表时间：
2012-06-01
期刊：
影响因子：
4.8
通讯作者：
Mechali, Marcel
中科院分区：
文献类型：
--
作者：
Cayrou, Christelle;Gregoire, Damien;Mechali, Marcel
了解后生动物 DNA 复制起点的本质是相当具有挑战性的。在缺乏像酵母那样的遗传测定的情况下，方法是基于 DNA 结构、在起始处合成的第一条新生链的可视化或定量，或起始结合蛋白的定位来设计的。 DNA 合成起始过程中 RNA 引发的新生 DNA 的纯化和定量是目前绘制活性复制起点最详尽、最精确的方法。我们已将该方法升级到通过微阵列或深度测序进行全基因组分析所需的重现性和富集水平。我们在这里详细介绍了不同步骤所需的协议和控制。 (c) 2012 年，爱思唯尔公司出版。
Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps. (c) 2012 Published by Elsevier Inc.