Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast
Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast
复制标题
DOI：
10.1038/s41596-019-0280-1
复制
发表时间：
2020-01-31
期刊：
影响因子：
14.8
通讯作者：
Larionov, Vladimir
中科院分区：
文献类型：
--
作者：
Kouprina, Natalay;Noskov, Vladimir N.;Larionov, Vladimir
在这里，我们描述了原始转化相关重组（TAR）克隆协议的扩展，能够从微生物基因组中选择性地分离DNA片段。该技术基于先前描述的TAR克隆程序，该程序开发用于从哺乳动物基因组中分离理想区域，该区域富含自主复制序列（ARS）样序列，这些序列是酵母中作为复制起源的元素。这样的序列在微生物基因组中并不常见。在本协议扩展中，将ARS与反选择标记一起插入到TAR载体中，允许选择克隆事件以防止载体循环。用CRISPR-Cas9预处理微生物DNA，在目标序列附近产生双链断裂，大大增加了区域阳性菌落的产量。与其他现有方法相比，该协议扩展允许从微生物基因组和环境DNA样本中选择性分离任何区域。整个过程可以在10 d内完成。在将TAR克隆应用于哺乳动物基因组的原始协议的扩展中，作者将该技术应用于微生物和环境DNA样本，通过将微生物基因组中不常见的ars样元素添加到TAR克隆载体中。
Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.In this extension to their original protocol applying TAR cloning to mammalian genomes, the authors apply the technique to microbes and environmental DNA samples, by adding ARS-like elements not commonly found in microbial genomes to the TAR cloning vector.