Siegel Chloe S, Stevenson Florence O, Zimmer Elizabeth A
Department of Crop Sciences: Plant Biotechnology and Molecular Biology, University of Illinois at Urbana-Champaign, 1102 S. Goodwin Avenue, Urbana, Illinois 61801 USA.
Department of Mechanical Engineering, Department of Mathematics, University of Maryland, 2181 Glenn L. Martin Hall, Building 088, College Park, Maryland 20742 USA.
Appl Plant Sci. 2017 Feb 15;5(2). doi: 10.3732/apps.1600109. eCollection 2017 Feb.
An efficient, effective DNA extraction method is necessary for comprehensive analysis of plant genomes. This study analyzed the quality of DNA obtained using paper FTA cards prepared directly in the field when compared to the more traditional cetyltrimethylammonium bromide (CTAB)-based extraction methods from silica-dried samples.
DNA was extracted using FTA cards according to the manufacturer's protocol. In parallel, CTAB-based extractions were done using the automated AutoGen DNA isolation system. DNA quality for both methods was determined for 15 non-agricultural species collected in situ, by gel separation, spectrophotometry, fluorometry, and successful amplification and sequencing of nuclear and chloroplast gene markers.
The FTA card extraction method yielded less concentrated, but also less fragmented samples than the CTAB-based technique. The card-extracted samples provided DNA that could be successfully amplified and sequenced. The FTA cards are also useful because the collected samples do not require refrigeration, extensive laboratory expertise, or as many hazardous chemicals as extractions using the CTAB-based technique.
The relative success of the FTA card method in our study suggested that this method could be a valuable tool for studies in plant population genetics and conservation biology that may involve screening of hundreds of individual plants. The FTA cards, like the silica gel samples, do not contain plant material capable of propagation, and therefore do not require permits from the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) for transportation.
对于植物基因组的全面分析而言,一种高效、有效的DNA提取方法是必要的。本研究分析了直接在野外制备的纸质FTA卡所获得的DNA质量,并将其与更传统的基于十六烷基三甲基溴化铵(CTAB)从硅胶干燥样品中提取DNA的方法进行了比较。
根据制造商的方案,使用FTA卡提取DNA。同时,使用自动AutoGen DNA分离系统进行基于CTAB的提取。通过凝胶分离、分光光度法、荧光法以及核基因和叶绿体基因标记的成功扩增与测序,对原位采集的15种非农业物种的两种方法的DNA质量进行了测定。
与基于CTAB的技术相比,FTA卡提取方法得到的样品浓度较低,但片段化程度也较低。卡提取的样品提供了能够成功扩增和测序的DNA。FTA卡也很有用,因为采集的样品不需要冷藏、不需要广泛的实验室专业知识,也不需要像基于CTAB技术的提取那样使用大量危险化学品。
FTA卡方法在我们研究中的相对成功表明,该方法对于可能涉及筛选数百株个体植物的植物群体遗传学和保护生物学研究可能是一种有价值的工具。与硅胶样品一样,FTA卡不包含能够繁殖的植物材料,因此运输时不需要获得美国农业部动植物卫生检验局(USDA-APHIS)的许可。