Purification of mitochondrial and plastid DNA
Purification of mitochondrial and plastid DNA
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DOI：
10.1038/nprot.2007.58
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Burger, Gertraud
中科院分区：
文献类型：
--
作者：
Lang, B. Franz;Burger, Gertraud
真核生物中线粒体和质体基因组的大小、结构和构象差异很大。同样，提取的细胞器 DNA 的产量和纯度也各不相同，它们是限制性图谱和测序实验成功的关键因素。我们在此描述了从多种真核生物中纯化细胞器 DNA 的程序。通过强调基本原则，这些程序将促进新的物种特异性协议的制定。所提出的纯化方案包括分离细胞器并随后从该亚细胞部分提取 DNA，或处理全细胞裂解物，然后进行 CsCl 梯度离心，根据 A + T 含量分离核 DNA 和细胞器 DNA。我们已经成功地使用所描述的程序对不同真核生物（包括非无菌原生生物）进行细胞器基因组测序。程序可在 3-5 天内完成，通常会产生几微克的 DNA 样本，用于对完整基因组进行测序。
The size, structure and conformation of mitochondrial and plastid genomes differ dramatically among eukaryotes. Similarly, the yield and purity of extracted organelle DNA also vary, and are crucial factors for the success of restriction mapping and sequencing experiments. We describe here procedures for the purification of organelle DNA from a broad range of eukaryotes. By emphasizing the underlying principles, these procedures will facilitate the development of new species-specific protocols. The presented purification schemes involve either isolation of organelles and subsequent extraction of DNA from this subcellular fraction, or processing of whole-cell lysates followed by CsCl gradient centrifugation to separate nuclear and organelle DNAs according to their A + T content. We have successfully used the described procedures for organelle genome sequencing from diverse eukaryotes, including non-axenic protists. Procedures can be completed in 3-5 days, typically yielding a few micrograms of DNA-ample for sequencing complete genomes.