Hept Megan A, Greene Lesley H
Department of Chemistry and Biochemistry, Old Dominion University, United States.
MethodsX. 2023 Aug 23;11:102341. doi: 10.1016/j.mex.2023.102341. eCollection 2023 Dec.
Extraction of high molecular weight (HMW) DNA for long read sequencing with little to no fragmentation and high purity is difficult to acquire from cyanobacterial species. Here we describe a modified method of extraction using Promega's Wizard HMW DNA Extraction Kit to acquire high molecular weight DNA from two cyanobacterial species. The protocol used in the kit is the "3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria" protocol. During a key step in the protocol, we propose that the lingering remnants of the cellular debris such as the mucilage layer of the cyanobacterial species is removed, preventing it from sticking to the DNA pellet produced. This customized modification is done between steps 11 and 12 and called METIS (maximizing extraction, transfer isopropanol step). This step drastically reduces the remaining mucilage layer, which if kept will stick to the DNA and make the DNA unsuitable for sensitive downstream next generation sequencing, like PacBio Sequencing. This protocol has been used to assemble two genomes from cyanobacteria ( and ) and one from a gram-negative bacterium, . It also allows for HMW DNA to be rapidly extracted without the use of toxic chemicals such as phenol and without extra reagents to be purchased.•Maximizing extraction, transfer isopropanol step (METIS) is the key modification during the step of DNA unraveling•METIS reduces leftover remnants of the mucilage layer in the extraction•High molecular weight DNA is produced with little to no fragmentation, and both a high purity and concentration.
从蓝藻物种中获取用于长读长测序的高分子量（HMW）DNA，且几乎没有片段化且纯度高，这很难实现。在此，我们描述一种使用Promega公司的Wizard HMW DNA提取试剂盒的改良提取方法，以从两种蓝藻物种中获取高分子量DNA。试剂盒中使用的方案是“3.D. 从革兰氏阳性和革兰氏阴性细菌中分离HMW DNA”方案。在该方案的一个关键步骤中，我们建议去除细胞碎片的残留部分，如蓝藻物种的黏液层，防止其黏附在产生的DNA沉淀上。这种定制的改良在步骤11和12之间进行，称为METIS（最大化提取，转移异丙醇步骤）。这一步骤大幅减少了残留的黏液层，如果不减少，黏液层会黏附在DNA上，使DNA不适用于像PacBio测序这样敏感的下游新一代测序。该方案已用于组装来自蓝藻（ 和 ）的两个基因组以及来自革兰氏阴性细菌 的一个基因组。它还允许在不使用苯酚等有毒化学物质且无需购买额外试剂的情况下快速提取HMW DNA。•最大化提取，转移异丙醇步骤（METIS）是DNA解链步骤中的关键改良•METIS减少了提取过程中黏液层的残留部分•产生的高分子量DNA几乎没有片段化，且具有高纯度和高浓度。
