Extracting single genomes from heterogenous DNA samples:: A test case with Carsonella ruddii, the bacterial symbiont of psyllids (Insecta) -: art. no. 3
Extracting single genomes from heterogenous DNA samples:: A test case with Carsonella ruddii, the bacterial symbiont of psyllids (Insecta) -: art. no. 3
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DOI：
10.1093/jis/5.1.3
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发表时间：
2005-03-16
影响因子：
2.2
通讯作者：
Ochman, H
中科院分区：
文献类型：
--
作者：
Dale, C;Dunbar, H;Ochman, H
由于无法从复杂的细胞混合物中分离出单个物种或无法在纯培养物中繁殖细胞，许多细菌基因组的分析受到阻碍。这个问题是研究许多生活在昆虫和其他动物细胞内的细菌共生体的障碍。为了从复杂的样品中回收细菌DNA，我们设计了一种方法，该方法有助于克隆具有独特G+C含量的DNA片段，以产生富含具有特定碱基组成的插入片段的鸟枪DNA文库。DNA制备物首先用在特定基因组中具有共同切割位点的限制酶处理，然后在大小分级分离后进行鸟枪克隆。这种方法适用于整个细菌组的木虱，Pachypsylla venusta，其中窝藏细菌共生体Carsonella ruddii。所得文库在细菌序列中高度富集。通过使用替代酶和部分酶，该技术可以适用于产生几乎纯的DNA文库的个别细菌物种。
Analysis of many bacterial genomes is impeded by the inability to separate individual species from complex mixtures of cells or to propagate cells in pure culture. This problem is an obstacle to the study of many bacterial symbionts that live intracellularly in insects and other animals. To recover bacterial DNA from complex samples, we devised a method that facilitates the cloning of DNA fragments of distinctive G+C contents in order to generate shotgun DNA libraries enriched in inserts having a specific base composition. DNA preparations are first treated with a restriction enzyme having a common cleavage site in a particular genome and then shotgun cloned following size-fractionation. This method was applied to whole bacteriomes of the psyllid, Pachypsylla venusta, which harbors the bacterial symbiont Candidatus Carsonella ruddii. The resulting libraries were highly enriched in bacterial sequences. Through the use of alternate enzymes and partial digests, this technique can be adapted to yield virtually pure DNA libraries for individual bacterial species.