Kuhn Ramona, Böllmann Jörg, Krahl Kathrin, Bryant Isaac Mbir, Martienssen Marion
Brandenburg University of Technology Cottbus-Senftenberg, Institute of Environmental Technology, 03046 Cottbus, Germany.
Brandenburg University of Technology Cottbus-Senftenberg, Institute of Environmental Technology, 03046 Cottbus, Germany.
J Microbiol Methods. 2017 Dec;143:78-86. doi: 10.1016/j.mimet.2017.10.007. Epub 2017 Oct 19.
DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen. In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established. Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits.
用于分子生物学应用的DNA提取通常需要根据样品来源优化提取程序。对于环境样品，已经开发了一系列不同的程序。我们使用四种不同的环境样品，即生活污水处理厂的活性污泥、河流沉积物、厌氧消化污泥和硝化富集培养物，比较了近期文献中发表的十种选定DNA提取方法的适用性和效率。我们基于DNA产量和质量评估了提取程序的适用性。在用PicoGreen染色后，通过紫外（UV）分光光度法和荧光分光光度法进行DNA定量。在我们的研究中，大多数情况下基于UV测量的DNA产量被高估，而荧光测量的DNA产量与粗DNA琼脂糖凝胶上的样品上样量相关性良好。通过粗DNA的凝胶电泳和使用通用引物对27f/1525r的16S rDNA的PCR产物来确定DNA提取物的质量。观察到粗DNA的凝胶电泳并不总是适合评估DNA的完整性和纯度，因为干扰背景物质（例如腐殖质）不可见。因此，当建立一种新的提取方法时，我们强烈建议通过凝胶电泳检查粗DNA和16S rDNA PCR产物的DNA质量。总之，我们发现十种提取程序中有四种适用于所有测试样品，没有明显限制。程序G（根据下萨克森州消费者保护和食品安全办公室的标准方法432_10401）比程序J（由Wilson于2001年发表）具有最广泛的应用范围。其次是程序F（Singka等人，2012年）和A（Bourrain等人，1999年）。所有这四种提取程序都能提供可靠且可重复的粗DNA和PCR产物。从经济角度来看，本研究中测试的所有程序都比商业DNA提取试剂盒便宜。
