In situ Detection of Specific DNA Double Strand Breaks using Rolling Circle Amplification
In situ Detection of Specific DNA Double Strand Breaks using Rolling Circle Amplification
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DOI：
10.4161/cc.4.12.2211
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发表时间：
2005-09
期刊：
影响因子：
4.3
通讯作者：
Jia Li;C. Young;Paul M. Lizardia;D. Stern
中科院分区：
文献类型：
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作者：
Jia Li;C. Young;Paul M. Lizardia;D. Stern
我们已经发展了一种方法，定位DNA双链断裂（DSB）原位在培养的哺乳动物细胞。用编码酿酒酵母HO内切酶及其切割位点的腺病毒诱导位点特异性DSB。滚环扩增（rolling circle amplification，RCA）是一种快速等温扩增检测单分子事件的灵敏方法，仅在同时感染编码HO内切酶和HO切割位点的腺病毒的细胞中观察到点状RCA信号，而在模拟感染或仅感染HO切割位点或内切酶病毒的细胞中均未观察到点状RCA信号。通过使用化学交联剂，原位RCA和免疫荧光（IF）可以在同一样品上同时进行。该方法为研究哺乳动物细胞中的DNA重组、DNA修复和检查点控制提供了一种新的方法。
We have developed a method to localize DNA double strand breaks (DSBs) insitu in cultured mammalian cells. Adenoviruses encoding Saccharomyces cerevisiae HOendonuclease and its cleavage site were used to induce site-specific DSBs. Rolling circleamplification (RCA), a sensitive method that allows the detection of single molecularevent by rapid isothermal amplification, was used to localize the broken ends in situ.Punctate RCA signals were only seen in the cells that had been infected with bothadenoviruses encoding HO endonuclease and HO cleavage site, but not in the cells mockinfectedor infected with the site or endonuclease virus only. With use of a chemicalcrosslinker, in situ RCA and immunofluorescence (IF) can be performed simultaneouslyon the same sample. This methodology provides a novel approach for investigation ofDNA recombination, DNA repair, and checkpoint controls in mammalian cells.