Sequencing complete mitochondrial and plastid genomes
Sequencing complete mitochondrial and plastid genomes
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DOI：
10.1038/nprot.2007.59
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Lang, B. Franz
中科院分区：
文献类型：
--
作者：
Burger, Gertraud;Lavrov, Dennis V.;Lang, B. Franz
细胞器基因组学已成为一个日益重要的研究领域，在分子建模、系统发育、分类学、群体遗传学和生物多样性方面都有应用。通常，研究项目涉及对完整的线粒体和质体基因组序列进行测定和比较分析，这些序列要么来自近缘物种，要么来自分类学上范围广泛的生物。在此，我们描述了两种可供选择的细胞器基因组测序方案。“随机基因组测序”方案适用于绝大多数细胞器基因组，无论其大小如何。它包括通过剪切（雾化）使DNA片段化，并将所得片段平端克隆到pUC或BlueScript类型的载体中。该方案在克隆文库的随机性以及时间和成本效益方面表现出色。“基于长PCR的基因组测序”方案专门适用于纯度和数量较低的DNA，对小的细胞器基因组特别有效。通过这两种方案中的任何一种构建文库都可在1周内完成。
Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The "random genome sequencing'' protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The "long-PCR-based genome sequencing'' protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week.