Specific single-cell isolation and genomic amplification of uncultured microorganisms
Specific single-cell isolation and genomic amplification of uncultured microorganisms
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DOI：
10.1007/s00253-006-0725-7
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发表时间：
2007-03-01
影响因子：
5
通讯作者：
Westermann, Peter
中科院分区：
文献类型：
--
作者：
Kvist, Thomas;Ahring, Birgitte K.;Westermann, Peter
我们在这项研究中描述了一种对单个未培养的原核生物进行基因组研究的新方法，该方法用于土壤古菌的分离和部分基因组测序。通过使用组特异性引物结合末端限制性片段长度多态性谱生成克隆文库，绘制了土壤样品中古细菌的多样性图谱。从环境样品中提取完整的细胞，然后使用从克隆库设计的 Cy3 标记探针进行荧光原位杂交探测来检测感兴趣的生物体。使用显微操作器分离具有明亮荧光信号的单个细胞，并将单个分离细胞的基因组用作使用 Phi29 DNA 聚合酶进行多重置换扩增 (MDA) 的模板。生成的 MDA 产物随后用于 16S rRNA 基因序列分析，并通过鸟枪法克隆进行额外的基因组分析。序列分析显示，与土壤泉古菌克隆 SCA1170 的 16S rRNA 基因同源性大于 99%，鸟枪片段与先前从土壤样品中检索到的泉古菌 BAC 克隆具有最接近的匹配。使用嗜热自养甲烷嗜热杆菌作为单细胞测试生物对该系统进行了验证，验证设置与该生物基因组的 10 个测试区域产生了 100% 的序列同源性。
We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed > 99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.