Nair Harisree P, Vincent Helvin, Bhat Sarita G
Department of Biotechnology, Cochin University of Science and Technology, Cochin, Kerala 682022, India.
Biotechnol Rep (Amst). 2014 Sep 30;4:134-138. doi: 10.1016/j.btre.2014.09.008. eCollection 2014 Dec.
Microbes in nature are rarely amenable to growth by standard microbiological methods, with the majority being unculturable. Metagenomic methods help to bypass and overcome the limitations of traditional culturing method; wherein total community DNA is isolated, cloned into suitable vector and host systems. However, isolation of total community DNA itself remains a challenge. In this study five methods of total community DNA isolation from three different mangrove soils were evaluated to test its PCR amenability. The yield and purity of the isolated DNA was also analysed. The quantity of DNA by all 5 methods although reasonably high, contained residual humic contaminants. Of the five, the method employing liquid nitrogen yielded readily amplifiable DNA, while that by all others required further downstream processing to achieve purity and PCR amenability.
自然界中的微生物很少能通过标准微生物学方法进行培养，大多数微生物无法培养。宏基因组学方法有助于绕过和克服传统培养方法的局限性；其中从整个群落中分离DNA，克隆到合适的载体和宿主系统中。然而，分离整个群落的DNA本身仍然是一项挑战。在本研究中，评估了从三种不同红树林土壤中分离总群落DNA的五种方法，以测试其对PCR的适用性。还分析了分离出的DNA的产量和纯度。尽管所有5种方法得到的DNA量都相当高，但都含有残留的腐殖质污染物。在这五种方法中，使用液氮的方法能产生易于扩增的DNA，而其他所有方法得到的DNA都需要进一步的下游处理以实现纯度和PCR适用性。
