Evaluation of DNA extraction methods for freshwater eukaryotic microalgae
Evaluation of DNA extraction methods for freshwater eukaryotic microalgae
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DOI：
10.1016/j.watres.2012.07.023
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发表时间：
2012-10-15
期刊：
影响因子：
12.8
通讯作者：
Mota, Cesar R.
中科院分区：
文献类型：
--
作者：
Eland, Lucy E.;Davenport, Russell;Mota, Cesar R.
使用分子方法研究天然和工程淡水资源的微藻群落尚处于起步阶段，之前的大多数研究都是通过显微镜进行的。微藻群落成员的低效或差异性 DNA 提取可能会导致下游群落分析出现偏差。三种市售 DNA 提取试剂盒已在一系列具有不同细胞壁的纯培养淡水藻类和取自富营养化废物稳定池 (WSP) 的混合藻类培养物上进行了测试。评估了 DNA 产量和质量，以及通过聚合酶链式反应 (PCR) 扩增 18S rRNA 基因片段的 DNA 适应性。 QiagenDNeasy (R) 血液和组织试剂盒 (QBT) 被发现可提供最高的 DNA 产量和质量。变性梯度凝胶电泳 (DGGE) 用于评估提取 DNA 的群落的多样性。在评估多样性时，试剂盒之间没有发现显着差异。建议将 QBT 与 WSP 样本一起使用，这一结论通过对两个热带 WSP 系统的群落进行进一步测试而得到证实。研究发现，在提取 DNA 之前用乙醇固定微藻样品会降低产量和多样性，因此不建议这样做。 (C) 2012 Elsevier Ltd. 保留所有权利。
The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy (R) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended. (C) 2012 Elsevier Ltd. All rights reserved.