Measurement of DNA synthesis and strand breaks using alkaline sucrose density gradient centrifugation.
Measurement of DNA synthesis and strand breaks using alkaline sucrose density gradient centrifugation.
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DOI：
10.1007/978-1-4020-4896-8_42
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发表时间：
2006
影响因子：
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通讯作者：
Kouich Yamada;J. Takezawa
中科院分区：
文献类型：
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作者：
Kouich Yamada;J. Takezawa
碱性蔗糖密度梯度（ASDG）离心可能是唯一的方法来检测“脉冲标记”的复制产物在细胞中的延伸。如果细胞在暴露于某些DNA-d损伤剂后被脉冲标记，则其“复制后修复”可通过ASDG技术来测量。对于未受损的细胞，也可以观察到复制子大小的正常复制。此外，该方法也适用于测量单链断裂。我们已经修改了这个经典的方法，可重复检测非常长的单链DNA在兆碱基水平。在此，该方案针对DT 40细胞进行了优化。
Alkaline sucrose density gradient (ASDG) centrifugation is probably an only method to detect elongation of “pulse-labeled “replication products in cells. If the cells are pulse-labeled after being exposed to some DNA-d damaging agents, their “post-replication repair” can be measured by ASDG technique. With non-damaged cells, normal replication in replicon size can be observed, too. In addition, the method is also applicable to measure single strand breaks. We have modified this classical method to reproducibly detect very long single-stranded DNA at the megabase level. Here, the protocols are optimized to DT40 cells.