Isolation of restriction fragments containing origins of replication from complex genomes.
Isolation of restriction fragments containing origins of replication from complex genomes.
复制标题
从复杂基因组中分离含有复制起点的限制性片段。
DOI：
10.1007/978-1-60327-815-7_17
复制
发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Hamlin,JoyceL
中科院分区：
文献类型：
--
作者：
Mesner,LarryD;Hamlin,JoyceL
由于哺乳动物基因组的高度复杂性，从这些基因组中鉴定和分离复制起点一直是一项艰巨的任务。然而，近年来已经改进了两种方法，每种方法都允许从异步细胞培养物中显著富集最近激活的复制起点。在其中一种方法中，新生链从长模板DNA中熔化，并且在蔗糖梯度上分离小的、以起源为中心的链。第二种方法涉及选择性截留的泡沫含有片段的凝胶琼脂糖和随后的回收和分离的分子克隆。通过这种方法从中国仓鼠和人类细胞制备的文库已被证明是非常纯的，并提供了一种可再生的来源，可用作微阵列上的探针或通过高通量技术测序以将其定位在基因组来源内。本文描述了用于异步哺乳动物细胞的气泡捕获方法，所述异步哺乳动物细胞以合理的倍增时间生长，并且可以可靠地制备核基质。制备核基质和富集复制中间体的方法描述于标题为“用于2-D凝胶分析的从哺乳动物细胞纯化含有复制中间体的限制性片段”的随附章节中。
The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently activated origins of replication from asynchronous cell cultures. In one of these, nascent strands are melted from the long template DNA, and the small, origin-centered strands are isolated on sucrose gradients. The second method involves the selective entrapment of bubble-containing fragments in gelling agarose and their subsequent recovery and isolation by molecular cloning. Libraries prepared by this method from Chinese hamster and human cells have been shown to be extremely pure, and provide a renewable resource of origins that can be used as probes on microarrays or sequenced by high-throughput techniques to localize them within the genomic source. The bubble-trapping method is described here for asynchronous mammalian cells that grow with reasonable doubling times and from which nuclear matrices can be reliably prepared. The method for nuclear matrix preparation and enrichment of replication intermediates is described in an accompanying chapter entitled, “Purification of Restriction Fragments Containing Replication Intermediates from Mammalian Cells for 2-D Gel Analysis”).