Epperson L Elaine, Strong Michael
Center for Genes, Environment, and Health, National Jewish Health, 1400 Jackson Street, Denver, Colorado 80206, USA.
J Clin Tuberc Other Mycobact Dis. 2020 Feb 4;19:100150. doi: 10.1016/j.jctube.2020.100150. eCollection 2020 May.
The rapid development in sequencing technology is creating an increase in demand for largely intact DNA as starting material as very long strands of DNA are sequenced directly to generate reads that are thousands of bases long. Organisms with thick cell walls are difficult to lyse, often impacting both DNA recovery and quality. Consequently, most mycobacterial DNA extraction methods require bead-beating steps or toxic chemicals. Here we present an updated method that yields abundant, high quality genomic DNA from and diverse nontuberculous mycobacterial (NTM) species, in addition to complex biological communities from a variety of sources. This method eliminates the time-consuming phenol and chloroform extraction and ethanol precipitation steps, and high quality DNA from up to 96 samples can be extracted in about 2-3 h of hands-on time. This DNA is suitable for long and short read sequencing technologies as well as PCR and qPCR amplification.
测序技术的快速发展使得对大量完整DNA作为起始材料的需求不断增加，因为非常长的DNA链会直接进行测序以生成数千个碱基长的读数。具有厚细胞壁的生物体难以裂解，这常常会影响DNA的回收率和质量。因此，大多数分枝杆菌DNA提取方法都需要珠磨步骤或有毒化学物质。在这里，我们提出了一种更新的方法，该方法除了可以从各种来源的复杂生物群落中提取外，还能从多种非结核分枝杆菌（NTM）物种中获得大量高质量的基因组DNA。该方法省去了耗时的苯酚和氯仿提取以及乙醇沉淀步骤，大约2-3小时的实际操作时间内即可从多达96个样品中提取高质量DNA。这种DNA适用于长读长和短读长测序技术以及PCR和qPCR扩增。
