A procedure for separate recovery of extra- and intracellular DNA from a single marine sediment sample
A procedure for separate recovery of extra- and intracellular DNA from a single marine sediment sample
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DOI：
10.1016/j.mimet.2014.06.009
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发表时间：
2014-09-01
影响因子：
2.2
通讯作者：
Kallmeyer, Jens
中科院分区：
文献类型：
--
作者：
Alawi, Mashal;Schneider, Beate;Kallmeyer, Jens
细胞外DNA （Extracellular DNA, eDNA）是水生沉积物和土壤中普遍存在的生物化合物。已有研究表明，eDNA在生物地球化学元素循环、基因水平转移和生物膜结构稳定等方面发挥着重要作用。以往的eDNA提取方法要么不适合贫营养沉积物，要么只能定量而不能进行遗传分析。我们的程序是基于细胞分离和eDNA从沉积物颗粒中解放，用碱性磷酸钠缓冲液连续洗涤，然后分离细胞和eDNA。然后将分离的eDNA结合到二氧化硅颗粒上并纯化，而从分离的细胞中提取细胞内DNA则使用商用试剂盒。该方法提供高纯度的细胞外和细胞内DNA，适用于PCR等下游应用。细胞外DNA是从波罗的海富含有机物的浅层沉积物、巴伦支海受冰川影响的沉积物和营养不良的南太平洋环流中提取的。这些样品中的eDNA浓度在23 ~ 626 ng g（-1）湿重沉积物中变化。为了验证每个处理步骤，进行了大量的实验。虽然提取效率高于其他已发表的方法，但它不是完全定量的。(C) 2014 Elsevier B.V.版权所有
Extracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Previous studies suggested that eDNA plays an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Previous methods for eDNA extraction were either not suitable for oligotrophic sediments or only allowed quantification but no genetic analyses. Our procedure is based on cell detachment and eDNA liberation from sediment particles by sequential washing with an alkaline sodium phosphate buffer followed by a separation of cells and eDNA. The separated eDNA is then bound onto silica particles and purified, whereas the intracellular DNA from the separated cells is extracted using a commercial kit. The method provides extra- and intracellular DNA of high purity that is suitable for downstream applications like PCR. Extracellular DNA was extracted from organic-rich shallow sediment of the Baltic Sea, glacially influenced sediment of the Barents Sea and from the oligotrophic South Pacific Gyre. The eDNA concentration in these samples varied from 23 to 626 ng g(-1) wet weight sediment. A number of experiments were performed to verify each processing step. Although extraction efficiency is higher than other published methods, it is not fully quantitative. (C) 2014 Elsevier B.V. All rights reserved.