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适用于高粘土含量酸性土壤的间接DNA提取方法。

Indirect DNA extraction method suitable for acidic soil with high clay content.

作者信息

Högfors-Rönnholm Eva, Christel Stephan, Engblom Sten, Dopson Mark

机构信息

Research and Development, Novia University of Applied Sciences, Vaasa, Finland.

Centre for Ecology and Evolution in Microbial Model Systems (EEMiS), Linnaeus University, Kalmar, Sweden.

出版信息

MethodsX. 2018 Feb 22;5:136-140. doi: 10.1016/j.mex.2018.02.005. eCollection 2018.

Abstract

DNA extraction is an essential procedure when investigating microbial communities in environmental samples by sequencing technologies. High clay soils can be problematic as DNA adsorbs to the clay particles and can thereby be preserved from lysed, non-viable cells for a substantial period of time. In order to accurately estimate the intact and living microbial community in the soil, extracellular DNA from dead, remnant bacterial cells needs to be removed prior to DNA extraction. One possibility is to use a sodium phosphate buffer to release both extracellular DNA and bacterial cells from the clay particles. After removing the extracellular DNA by centrifugation, the remaining viable cells can be harvested and DNA extracted. The described method is a modification of a procedure for separating extracellular DNA and bacterial cells from acidic clay soils. •The modified method increases bacterial cell yields from acidic clay soils, such as acid sulfate soil.•The modified method eliminates some steps from the original method, as only DNA from intact bacterial cells is required.•The indirect DNA extraction method increases the workload compared to standard direct extraction methods, but subsequent downstream analyses will give a more representative picture of the viable microbial community composition in the soil.

摘要

当通过测序技术研究环境样本中的微生物群落时,DNA提取是一个必不可少的步骤。高黏土土壤可能会带来问题,因为DNA会吸附到黏土颗粒上,从而可以在相当长的一段时间内从裂解的、无活力的细胞中保存下来。为了准确估计土壤中完整且有活力的微生物群落,在DNA提取之前需要去除死亡的残留细菌细胞中的细胞外DNA。一种可能性是使用磷酸钠缓冲液从黏土颗粒中释放细胞外DNA和细菌细胞。通过离心去除细胞外DNA后,可以收获剩余的有活力细胞并提取DNA。所描述的方法是一种从酸性黏土土壤中分离细胞外DNA和细菌细胞的方法的改进。•改进后的方法提高了从酸性黏土土壤(如酸性硫酸盐土壤)中获得的细菌细胞产量。•改进后的方法省去了原始方法中的一些步骤,因为只需要完整细菌细胞的DNA。•与标准直接提取方法相比,间接DNA提取方法增加了工作量,但后续的下游分析将更能代表土壤中有活力的微生物群落组成情况。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b480/6314954/828158d5abae/fx1.jpg

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