快速从哺乳动物细胞中提取线粒体 DNA 用于下一代测序。
Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing.
机构信息
Albert Einstein College of Medicine, Department of Genetics, New York, NY, USA.
出版信息
Biotechniques. 2013 Sep;55(3):133-6. doi: 10.2144/000114077.
Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequencing results. Here, we describe a fast, cost-effective, and reliable method for preparation of mtDNA enriched samples from eukaryotic cells ready for direct sequencing. Our protocol utilizes a conventional miniprep kit, paramagnetic bead-based purification, and an optional, limited PCR amplification of mtDNA. The first two steps alone provide more than 2000-fold enrichment for mtDNA when compared with total cellular DNA (~200-fold in comparison with current commercially available kits) as demonstrated by real-time PCR. The percentage of sequencing reads aligned to mtDNA was about 22% for non-amplified samples and greater than 99% for samples subjected to 10 cycles of long-range-PCR with mtDNA specific primers.
标准的线粒体 DNA（mtDNA）提取方法不能提供足够高的 mtDNA 富集度，无法直接进行测序，必须进行长距离 PCR 扩增，这可能会导致测序结果产生偏差。在这里，我们描述了一种从真核细胞中制备 mtDNA 富集样品的快速、经济高效且可靠的方法，该方法可直接用于测序。我们的方案利用了常规的小量制备试剂盒、基于磁性珠的纯化以及 mtDNA 的可选、有限 PCR 扩增。与总细胞 DNA 相比（与当前市售试剂盒相比约 200 倍），仅前两个步骤就可使 mtDNA 得到超过 2000 倍的富集（与当前市售试剂盒相比约 200 倍），这一点通过实时 PCR 得到了证明。对于未经扩增的样本，测序reads 与 mtDNA 比对的百分比约为 22%，而对于用 mtDNA 特异性引物进行 10 个循环长距离 PCR 的样本，该百分比大于 99%。
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