Institute of Human Genetics, CNRS, Montpellier 34396, France.
Methods. 2012 Jun;57(2):158-64. doi: 10.1016/j.ymeth.2012.06.015. Epub 2012 Jul 13.
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.
解析真核生物 DNA 复制起始点的性质极具挑战性。在缺乏像酵母那样的遗传检测方法的情况下，人们根据 DNA 结构、对在起始点合成的最初新生链的可视化或定量、或根据起始点结合蛋白的定位，设计了一些方法。在 DNA 合成起始时，通过纯化和定量 RNA 引发的新生 DNA，是目前为止在全基因组范围内进行分析以绘制活性复制起始点的最全面和精确的方法。我们已经将该方法的重复性和富集度提升到了微阵列或深度测序全基因组分析所需的水平。我们在这里详细介绍了不同步骤所需的方案和对照。
