Solomon Solly, Kachiprath Bhavya, Jayanath G, Sajeevan T P, Bright Singh I S, Philip Rosamma
Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Kochi, 16, Kerala, India.
National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, 16, Kerala, India.
3 Biotech. 2016 Dec;6(2):160. doi: 10.1007/s13205-016-0482-y. Epub 2016 Aug 6.
Recent advances in culture-independent studies of microbes had proved to be more reliable and efficient than the conventional ones. The isolation of good quality and quantity of total community DNA are one of the major hurdles in this endeavour. Shearing of DNA during the extraction process and the co-extraction of inhibitory compounds reduce the quality of the isolated nucleic acids making it unsuitable for the construction of large insert metagenomic libraries. In the present study, a multi-level filtration step was brought in which efficiently isolated total bacterial DNA from three different environment samples. The preprocessing method could efficiently improve the 260/230 ratio of the isolated DNA by 2.3-45 % and decreased the protein contamination by 22.5-34.5 % on saltpan and arctic sediment samples, respectively. The more significant part of the experiment was that the DNA obtained was of high quality with minimal shearing making it most suitable for the construction of large insert genomic libraries. PCR amplification of 16S rRNA gene confirmed that the filtration method was effective in the isolation of high-quality DNA.
近年来，与传统方法相比，不依赖培养的微生物研究进展已被证明更可靠、更高效。获取高质量和足够数量的总群落DNA是这一研究中的主要障碍之一。DNA在提取过程中发生剪切，以及共提取的抑制性化合物，都会降低分离出的核酸质量，使其不适用于构建大插入片段宏基因组文库。在本研究中，引入了一个多级过滤步骤，可从三种不同环境样品中有效分离出总细菌DNA。该预处理方法可有效提高分离出的DNA的260/230比值，在盐田和北极沉积物样品中分别提高2.3% - 45%，并将蛋白质污染分别降低22.5% - 34.5%。该实验更重要的一点是，获得的DNA质量高，剪切极少，最适合构建大插入片段基因组文库。16S rRNA基因的PCR扩增证实，该过滤方法在分离高质量DNA方面是有效的。
