A chemical method for fast and sensitive detection of DNA synthesis in vivo
A chemical method for fast and sensitive detection of DNA synthesis in vivo
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DOI：
10.1073/pnas.0712168105
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发表时间：
2008-02-19
影响因子：
11.1
通讯作者：
Mitchison, Timothy J.
中科院分区：
文献类型：
--
作者：
Salic, Adrian;Mitchison, Timothy J.
我们开发了一种检测增殖细胞中DNA合成的方法，该方法基于5-乙炔基-2‘-脱氧尿苷(EDU)的掺入，并随后通过铜(I)催化的[3+2]环加成反应(“点击”化学)由荧光叠氮化合物检测。EDU标签的检测非常灵敏，可以在几分钟内完成。用于检测的荧光叠氮化物的小尺寸导致了高度的样品穿透，允许对大组织和器官外植体的整装制剂进行染色。与BrdU相比，该方法不需要样品固定或DNA变性，并且允许良好的结构保存。我们在培养的细胞以及整个动物的肠道和脑中演示了该方法的使用。
We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2 '-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.