Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification
Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification
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DOI：
10.1038/nprot.2014.031
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发表时间：
2014-03-01
期刊：
影响因子：
14.8
通讯作者：
Trouche, Didier
中科院分区：
文献类型：
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作者：
Chailleux, Catherine;Aymard, Francois;Trouche, Didier
我们对DNA双链断裂（DSB）的管理和修复的理解的最新进展依赖于对活细胞中由位点特异性内切酶（通常是重组限制性内切酶）的受控活性诱导的靶向DSB的研究。在这里，我们描述了一个协议，用于量化这些核酸内切酶诱导的DSB，这种量化是必不可少的DSB如何管理和修复的解释。将生物素化的双链寡核苷酸连接至酶切的基因组DNA，从而允许在链霉亲和素珠上纯化切割的DNA。然后通过定量PCR（qPCR）在给定位点或通过全基因组技术（例如，微阵列或高通量测序）。该技术称为连接介导的纯化，可在2d内完成。它比现有的替代方法更准确和灵敏，并且与全基因组分析兼容。它允许核酸内切酶介导的断裂量在两种条件之间或在整个基因组中进行精确比较，从而深入了解给定因素或各种染色质背景对局部修复参数的影响。
Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.