Mavrodiev Evgeny V, Dervinis Christopher, Whitten William Mark, Gitzendanner Matthew A, Kirst Matias, Kim Sangtae, Kinser Taliesin J, Soltis Pamela S, Soltis Douglas E
Florida Museum of Natural History University of Florida Gainesville Florida 32611 USA.
School of Forest Resources and Conservation University of Florida Gainesville Florida 32611 USA.
Appl Plant Sci. 2021 Apr 7;9(3):e11413. doi: 10.1002/aps3.11413. eCollection 2021 Mar.
Commonly used molecular techniques such as next-generation sequencing require reliable methods to extract DNA quickly and efficiently. Secondary compounds within plant tissues make this requirement all the more challenging, often forcing researchers to test different extraction methods tailored to their particular species of interest in order to obtain large amounts of high-quality genomic DNA. The opportunities provided by high-throughput, next-generation sequencing only exacerbate these problems, especially when trying to extract DNA from multiple species at the same time. Several methods have attempted to resolve the challenges of obtaining suitable DNA from plants; however, a rapid, high-yield, high-quality, and highly scalable DNA extraction method is still needed.
We present a rapid DNA extraction protocol that utilizes a buffer with relatively large amounts of cetyltrimethylammonium bromide (CTAB) and sodium chloride, combined with a silica maxi-column cleanup of the extracted DNA. The new method is easy to implement using standard equipment and inexpensive reagents. The entire procedure (from grinding to the final elution) can be completed in less than two hours for a single sample and can be easily scaled to meet desired research goals. It works on diverse green plants with highly varied secondary chemistries (e.g., ferns, gymnosperms, and phylogenetically divergent angiosperms).
Application of the protocol to various plant species yielded DNA of high quality in less than two hours and can be adjusted to extract DNA at large (maxi-preps) or small (96-well minipreps) scales. We anticipate that our method will be of wide utility for rapidly isolating large quantities of quality genomic DNA from diverse plant species and will have broad applications in phylogenetic studies utilizing PCR and short-read DNA sequencing.
常用的分子技术，如下一代测序，需要可靠的方法来快速高效地提取DNA。植物组织中的次生化合物使这一要求更具挑战性，这常常迫使研究人员测试针对其特定感兴趣物种量身定制的不同提取方法，以获得大量高质量的基因组DNA。高通量的下一代测序所带来的机遇只会加剧这些问题，尤其是在试图同时从多个物种中提取DNA时。已经有几种方法试图解决从植物中获得合适DNA的挑战；然而，仍然需要一种快速、高产、高质量且高度可扩展的DNA提取方法。
我们提出了一种快速DNA提取方案，该方案使用含有相对大量十六烷基三甲基溴化铵（CTAB）和氯化钠的缓冲液，并结合硅胶大柱对提取的DNA进行纯化。这种新方法使用标准设备和廉价试剂很容易实施。对于单个样品，整个过程（从研磨到最终洗脱）可以在不到两小时内完成，并且可以轻松扩展以满足所需的研究目标。它适用于次生化学性质差异很大的各种绿色植物（例如蕨类植物、裸子植物和系统发育上不同的被子植物）。
将该方案应用于各种植物物种，在不到两小时内就能获得高质量的DNA，并且可以调整以大规模（大制备）或小规模（96孔微量制备）提取DNA。我们预计，我们的方法将广泛用于从各种植物物种中快速分离大量高质量的基因组DNA，并将在利用PCR和短读长DNA测序的系统发育研究中有广泛应用。
