Analysis of Mitochondrial DNA by Two-Dimensional Agarose Gel Electrophoresis
Analysis of Mitochondrial DNA by Two-Dimensional Agarose Gel Electrophoresis
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DOI：
10.1007/978-1-59745-521-3_2
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发表时间：
2009-01-01
期刊：
影响因子：
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通讯作者：
Holt, Ian J.
中科院分区：
文献类型：
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作者：
Reyes, Aurelio;Yasukawa, Takehiro;Holt, Ian J.
在高等脊椎动物中，线粒体的DNA以大约16kbp的圆形分子的形式存在。这些圆以多基因组核蛋白复合物或类核排列。据设想，类核上层结构对mtDNA的复制和分离的双重过程作出了关键贡献。复制中间体可以从细胞或固体组织中分离出来，并在二维上分离油脂琼脂糖凝胶，从而揭示DNA复制机制的丰富数据。利用这项技术，我们已经证明，在高等脊椎动物中，许多复制mtDNA的分子具有广泛的RNA: DNA杂交区域。最近，我们提取了线粒体核蛋白，并通过同样的方法对其进行分析，以获得DNA结合蛋白在线粒体DNA上分布的信息。在这里，我们描述了从高等脊椎动物的肝脏和培养细胞中分离完整线粒体复制中间体的过程，以及在中性二维琼脂糖凝胶上分离DNA片段的过程。
In higher vertebrates, the DNA of mitochondria takes the form of circular molecules of approximately 16 kbp. These circles arc arranged in multigenomic nucleoprotein complexes or nucleoids. It is envisaged that nucleoid superstructure makes a critical contribution to the twin processes of replication and segregation of mtDNA. Replication intermediates can be isolated from cells or solid tissues and separated oil agarose gels in two dimensions to reveal a wealth of data on mechanisms of DNA replication. Using this technique we have demonstrated that many molecules of replicating mtDNA have extensive regions of RNA: DNA hybrid in higher vertebrates. More recently, we have extracted mitochondrial nucleoprotein and analyzed it by the same method to derive information oil the distribution of DNA-binding proteins on mitochondrial DNA. Here we describe the procedures used to isolate intact mitochondrial replication intermediates from liver and Cultured cells of higher vertebrates and the process of separating DNA fragments on neutral two-dimensional agarose gels.