一种用于体内DNA合成快速灵敏检测的化学方法。
A chemical method for fast and sensitive detection of DNA synthesis in vivo.
作者信息
Salic Adrian, Mitchison Timothy J
机构信息
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
出版信息
Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2415-20. doi: 10.1073/pnas.0712168105. Epub 2008 Feb 12.
Abstract
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.
摘要
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，并通过铜（I）催化的[3 + 2]环加成反应（“点击”化学），利用荧光叠氮化物对其进行后续检测。EdU标记的检测灵敏度很高，几分钟内即可完成。用于检测的荧光叠氮化物体积小，能高度穿透标本，从而可以对大型组织和器官外植体的整装制剂进行染色。与溴脱氧尿苷（BrdU）不同，该方法不需要样品固定或DNA变性，并且能很好地保持结构。我们展示了该方法在培养细胞以及完整动物的肠道和大脑中的应用。
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