Dale Colin, Dunbar Helen, Moran Nancy A, Ochman Howard
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
J Insect Sci. 2005;5:3. doi: 10.1093/jis/5.1.3.
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.
由于无法从复杂的细胞混合物中分离出单个物种或在纯培养物中培养细胞，许多细菌基因组的分析受到阻碍。这个问题是研究许多在昆虫和其他动物细胞内生活的细菌共生体的障碍。为了从复杂样品中回收细菌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，以便生成富含具有特定碱基组成插入片段的鸟枪法DNA文库。首先用在特定基因组中具有共同切割位点的限制性酶处理DNA制剂，然后在大小分级后进行鸟枪法克隆。该方法应用于携带细菌共生体鲁氏嗜菌菌（Candidatus Carsonella ruddii）的木虱（Pachypsylla venusta）的整个菌胞。所得文库高度富集细菌序列。通过使用替代酶和部分消化，该技术可适用于为单个细菌物种产生几乎纯的DNA文库。
