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Author:
Date:
2013-06-20
[Abstract] Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations of 3[H]-thymidine, possibly because they lack a high-affinity transporter for thymidine. Therefore, BrdU, a thymidine analog, has been used at high concentrations to study kDNA replication. However, the detection of BrdU with anti-BrdU antibody requires harsh conditions such as the acid or heat treatment to seperate double DNA strand, which affects the ability for other antibodies to bind as well as the morphology and ability for dyes that require dsDNA to bind efficiently. ...
[摘要] 锥虫体线粒体基因组，称为Kinetoplast DNA（kDNA），是互锁DNA环的大块网络。 kDNA复制和结构的研究具有重要意义，因为kDNA是有效的药物靶标。然而，在循环锥虫中的DNA不能用示踪剂浓度的3 H] - 胸苷标记，可能是因为它们缺少用于胸苷的高亲和性转运蛋白。因此，已经以高浓度使用BrdU（胸苷类似物）来研究kDNA复制。然而，用抗BrdU抗体检测BrdU需要苛刻的条件，例如分离双DNA链的酸或热处理，其影响其它抗体结合的能力以及需要dsDNA结合的染料的形态和能力有效率的。相反，EdU（5-乙炔基-2'-脱氧尿苷），一种新的胸苷类似物，可以用于研究kDNA复制和细胞增殖与简化的协议。 EdU的检测基于点击反应，其是叠氮化物和炔之间的铜（I）催化反应。这种点击反应不需要DNA变性，并且是多重兼容的，例如用于细胞周期分析的其他抗体和染料。为了显现锥虫复制的核DNA和kDNA，将EdU加入细胞培养基的培养基中，孵育0.5小时至3小时，然后通过以下程序检测。
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