Environmental Systems, University of California, 5200 North Lake Road, Merced, CA, 95343, USA.
Quantitative and Systems Biology, University of California, 5200 North Lake Road, Merced, CA, 95343, USA.
Mol Ecol Resour. 2017 Jul;17(4):721-729. doi: 10.1111/1755-0998.12620. Epub 2016 Nov 16.
The inclusion of next-generation sequencing technologies in population genetic and phylogenetic studies has elevated the need to balance time and cost of DNA extraction without compromising DNA quality. We tested eight extraction methods - ranging from low- to high-throughput techniques - and eight phyla: Annelida, Arthropoda, Cnidaria, Chordata, Echinodermata, Mollusca, Ochrophyta and Porifera. We assessed DNA yield, purity, efficacy and cost of each method. Extraction efficacy was quantified using the proportion of successful polymerase chain reaction (PCR) amplification of two molecular markers for metazoans (mitochondrial COI and nuclear histone 3) and one for Ochrophyta (mitochondrial nad6) at four time points - 0.5, 1, 2 and 3 years following extraction. DNA yield and purity were quantified using NanoDrop absorbance ratios. Cost was estimated in terms of time and material expense. Results show differences in DNA yield, purity and PCR success between extraction methods and that performance also varied by taxon. The traditional time-intensive, low-throughput CTAB phenol-chloroform extraction performed well across taxa, but other methods also performed well and provide the opportunity to reduce time spent at the bench and increase throughput.
下一代测序技术在群体遗传学和系统发育学研究中的应用，提高了在不影响 DNA 质量的前提下平衡 DNA 提取时间和成本的需求。我们测试了 8 种提取方法 - 从低通量技术到高通量技术 - 和 8 个门：环节动物门、节肢动物门、刺胞动物门、脊索动物门、棘皮动物门、软体动物门、黄藻门和多孔动物门。我们评估了每种方法的 DNA 产量、纯度、效率和成本。通过对两种后生动物分子标记（线粒体 COI 和核组蛋白 3）和一种黄藻（线粒体 nad6）的聚合酶链反应（PCR）扩增的成功比例，在提取后 0.5、1、2 和 3 年的四个时间点，定量评估了提取效率。使用 NanoDrop 吸光度比值定量 DNA 产量和纯度。以时间和材料费用来估计成本。结果表明，不同的提取方法在 DNA 产量、纯度和 PCR 成功率方面存在差异，而且性能也因分类群而异。传统的费时、低通量 CTAB 酚-氯仿提取方法在所有分类群中表现良好，但其他方法也表现良好，有机会减少在实验台上花费的时间并提高通量。
