Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing
Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing
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DOI：
10.1016/j.ymeth.2012.04.007
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发表时间：
2012-06-01
期刊：
影响因子：
4.8
通讯作者：
Pasero, Philippe
中科院分区：
文献类型：
--
作者：
Bianco, Julien N.;Poli, Jerome;Pasero, Philippe
DNA梳理是本西蒙及其同事开发的一种强大的方法，可以在硅化玻璃盖上拉伸DNA分子。该技术提供了一种独特的方法，在新合成的DNA中加入胸腺嘧啶类似物，如5-溴-2'-脱氧尿嘧啶（BrdU）、5-碘-2'-脱氧尿嘧啶（IdU）和5-氯-2'-脱氧尿嘧啶（CldU）后，在单个DNA分子水平上监测复制起始点的激活和复制分支的进展。与基于微阵列的方法不同，该分析可以获得单个细胞中复制谱的可变性。它还可用于监测DNA损伤对分叉进展、停止和重新启动的影响。在这篇综述中，我们提出了标准的DNA梳理方法来分析出芽酵母和人类细胞中的DNA复制。我们还表明，5-乙基-2'-脱氧尿嘧啶（EdU）可以作为BrdU的良好替代品用于DNA梳理分析，因为与卤化核苷酸不同，它可以在不事先变性DNA的情况下被检测到。(c) 2012 Elsevier Inc.版权所有。
DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA. (c) 2012 Elsevier Inc. All rights reserved.