Lienhard Andrea, Schäffer Sylvia
Institute of Biology, University of Graz, Graz, Austria.
PeerJ. 2019 Apr 12;7:e6753. doi: 10.7717/peerj.6753. eCollection 2019.
The application of an appropriate extraction method is a relevant factor for the success of all molecular studies.
Seven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated.
Although low DNA concentrations were measured for all methods, the results showed that-with the exception of two methods-the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results.
In conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations.
采用合适的提取方法是所有分子研究取得成功的一个重要因素。
通过应用九种不同方案，比较了七种适用于极小型节肢动物高通量DNA测序的不同DNA提取方法：三种基于硅胶的离心柱法、两种基于十六烷基三甲基溴化铵（CTAB）的方法（一种带有额外硅胶膜）、一种蛋白质沉淀法和一种基于螯合树脂的方法（应用不同方案）。对这些方法提取的DNA的数量（浓度）和质量（降解、污染、聚合酶链反应（PCR）及测序成功率）以及成本、制备时间、用户友好性和所需耗材进行了比较。为评估DNA数量，应用了两种不同的DNA浓度测量方法。此外，还评估了不同起始材料量（不同体型）、可变裂解温度以及DNA提取过程中混合操作的影响。
尽管所有方法测得的DNA浓度都较低，但结果表明，除两种方法外，PCR成功率为100%。然而，其他参数显示出巨大差异。进行DNA提取所需时间从20分钟到2.5小时不等（Chelex法与CTAB法），每个样本的成本从0.02欧元到3.46欧元不等（Chelex法与QIAamp试剂盒）。只有四种方法获得了高质量的基因组DNA。DNA数量测量结果进一步表明，一些仪器无法处理少量DNA，结果存在差异。
总之，结果表明使用Chelex（螯合树脂）是一种快速、低成本的方法，可为不同类型的分子研究提供高质量DNA。
