利用 5-乙炔基-2'-脱氧胞苷在基因组 DNA 中检测胞嘧啶掺入的细胞内情况。
Intracellular detection of cytosine incorporation in genomic DNA by using 5-ethynyl-2'-deoxycytidine.
机构信息
Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
出版信息
Chembiochem. 2011 Sep 19;12(14):2184-90. doi: 10.1002/cbic.201100353. Epub 2011 Aug 1.
Abstract
5-Ethynyl-2'-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo(-) and DNA polymerase β. Incorporation of 5-ethynyl-2'-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.
摘要
5-乙炔基-2'-脱氧胞苷三磷酸（EdCTP）被合成为一种探针，与荧光标记结合使用，以促进以 DNA 为中心的过程（DNA 复制、修复和胞嘧啶去甲基化）的体内动力学分析。动力学分析表明，EdCTP 被 Klenow exo(-) 和 DNA 聚合酶 β 接受为底物。这些酶将 5-乙炔基-2'-脱氧胞苷（EdC）掺入 DNA 的效率至多略低于天然 dC。通过使用带有荧光叠氮化物的点击反应，在聚合酶掺入和 T4 DNA 连接酶介导的连接后，观察到含有 EdC 的 DNA。随后在雄性小鼠生殖细胞和受精卵中的实验表明，EdC 是体内 DNA 复制的一种特异性和可靠的报告基因。
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