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Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast.
Nature Protocols ( IF 14.8 ) Pub Date : 2020-01-31 , DOI: 10.1038/s41596-019-0280-1 Natalay Kouprina 1 , Vladimir N Noskov 1 , Vladimir Larionov 1
Nature Protocols ( IF 14.8 ) Pub Date : 2020-01-31 , DOI: 10.1038/s41596-019-0280-1 Natalay Kouprina 1 , Vladimir N Noskov 1 , Vladimir Larionov 1
Affiliation
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.
中文翻译:
使用酵母中的转化相关重组克隆从单个微生物基因组和环境DNA样品中选择性分离大片段。
在这里,我们描述了我们原始的转化相关重组(TAR)克隆协议的扩展,可以从微生物基因组中选择性分离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出所需区域,该基因组富含自动复制序列(ARS)样序列,这些元件起着酵母中复制起点的作用。这样的序列在微生物基因组中并不常见。在此协议扩展中,将ARS与可逆选择标记一起插入TAR向量,从而可以选择针对向量环化的克隆事件。用CRISPR-Cas9对微生物DNA进行预处理以在目标序列附近产生双链断裂,大大提高了区域阳性菌落的产量。与其他可用方法相比,此协议扩展允许从微生物基因组以及环境DNA样品中选择性分离任何区域。整个过程可以在10天内完成。
更新日期:2020-01-31
中文翻译:
使用酵母中的转化相关重组克隆从单个微生物基因组和环境DNA样品中选择性分离大片段。
在这里,我们描述了我们原始的转化相关重组(TAR)克隆协议的扩展,可以从微生物基因组中选择性分离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出所需区域,该基因组富含自动复制序列(ARS)样序列,这些元件起着酵母中复制起点的作用。这样的序列在微生物基因组中并不常见。在此协议扩展中,将ARS与可逆选择标记一起插入TAR向量,从而可以选择针对向量环化的克隆事件。用CRISPR-Cas9对微生物DNA进行预处理以在目标序列附近产生双链断裂,大大提高了区域阳性菌落的产量。与其他可用方法相比,此协议扩展允许从微生物基因组以及环境DNA样品中选择性分离任何区域。整个过程可以在10天内完成。