Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India.
Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, 784028, Assam, India.
J Microbiol Methods. 2014 Feb;97:68-73. doi: 10.1016/j.mimet.2013.11.008. Epub 2013 Nov 23.
Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23 kb and yield 0.5-5 μg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction.
从土壤样本中提取 DNA 采用标准方法通常会导致产量低和质量差，由于较小模板 DNA 的嵌合产物的形成以及腐殖质的存在，因此不适合通过聚合酶链反应 (PCR) 进行群落分析。本研究基于处理时间、纯度、DNA 产量、PCR 适用性、限制性消化和 mDNA 文库构建，重点评估了从土壤样本中提取宏基因组 DNA 的五种不同方法。开发了一种简单快速的基于碱裂解的方法，该方法通过从土壤中间接提取 DNA 可以去除 90%的腐殖质，而不会剪切 DNA，并允许快速有效地分离高质量的 DNA，而无需十六烷基三甲基溴化铵和苯酚清洗。粗提物中的 DNA 片段大小>23kb，产量为 0.5-5μg/g 土壤。使用 Sephadex G-50 树脂对 mDNA 进行纯化，可以从土壤样品中获得高浓度的 DNA，已成功用于基于 16S rDNA 的扩增，使用 27F 和 1492R 通用引物扩增 1500bp 的 DNA 片段，然后进行限制性消化和 mDNA 文库构建。
