环境DNA(eDNA)工作流程包含许多熟悉的分子实验室技术,但也采用了几种独特的方法。当使用eDNA时,必须通过保存从收集点避免污染,并选择有意义的阴性对照。由于eDNA可以从各种样品和栖息地获得(例如,土壤,水,空气,或组织),协议将根据使用情况而有所不同。样品可能需要额外的步骤来稀释,块,或去除抑制剂或物理分解样品或过滤器。此后,采用标准DNA分离技术(基于试剂盒或苯酚:氯仿:异戊基[PCI])。一旦DNA被提取出来,它通常使用荧光计进行定量。收益率差异很大,但重要的是在扩增感兴趣的基因之前知道。鼓励采样材料和提取的DNA的长期储存,因为它为溢出/污染的样品提供了备份,数据丢失,重新分析,以及使用较新技术的未来研究。在冰箱中储存通常是理想的;然而,一些存储缓冲区(例如,Longmires)要求过滤器或拭子保持在室温下,以防止与缓冲液相关的溶质沉淀。这些eDNA分离的基线方法,验证,和保存在本协议章节中详细介绍。此外,我们概述了一个具有成本效益的,优化了自制提取协议以提取eDNA。
Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.