PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in Escherichia coli.
PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in Escherichia coli.
复制标题
基于 PCR 的完整小鼠线粒体基因组克隆和大肠杆菌中的稳定工程。
DOI：
10.1007/s10529-009-0063-9
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发表时间：
2009
影响因子：
2.7
通讯作者：
Koob,MichaelD
中科院分区：
文献类型：
--
作者：
Yoon,YoungGeol;Yang,Yi-Wei;Koob,MichaelD
我们设计了一种利用PCR扩增和顺序连接技术克隆哺乳动物线粒体基因组（mtDNA）的方法。在这里，我们通过克隆完整的小鼠mtDNA来测试这种方法。利用特异性限制性内切酶将线粒体DNA分为4 ~ 5个片段，用高保真长链DNA聚合酶进行扩增。将合成的片段单独克隆以测试它们在大肠杆菌宿主中的毒性，然后依次组合到含有大肠杆菌R6 K DNA复制起点的载体中。当维持在适度低的拷贝数/细胞时，合成的完整小鼠mtDNA克隆在大肠杆菌中稳定而忠实地复制。通过核苷酸序列测定证实了小鼠线粒体DNA克隆的序列完整性，基因组中没有发现突变或重排，这种方法可以促进在大肠杆菌中克隆整个哺乳动物线粒体基因组，并有助于将所需的修饰引入线粒体基因组。
We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) inEscherichiacoliusing PCR-based amplification and sequential ligation. Here we test this approach by cloning the complete mouse mtDNA. The mtDNA was divided into four to five fragments based on unique restriction enzyme sites and amplified by high-fidelity long-range DNA polymerase. The synthesized fragments were cloned individually to test their toxicity in theE.colihost and then combined sequentially into a vector containing theE.coliR6K origin of DNA replication. The synthetic complete mouse mtDNA clones were replicated stably and faithfully inE.coliwhen maintained at moderately low copy numbers per cell. The sequence integrity of the synthetic mouse mtDNA clones was confirmed by nucleotide sequencing; no mutations or rearrangements in the genome were found. This approach can facilitate the cloning of entire mammalian mitochondrial genomes inE.coliand assist in the introduction of desired modifications into the mitochondrial genome.