Webster Gordon, Newberry Carole J, Fry John C, Weightman Andrew J
Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, P.O. Box 915, CF10 3TL, S. Wales, Cardiff, UK.
J Microbiol Methods. 2003 Oct;55(1):155-64. doi: 10.1016/s0167-7012(03)00140-4.
Investigations into the deep marine environment have demonstrated the presence of a significant microbial biomass buried deep within sediments on a global scale. It is now believed that this deep biosphere plays a major role in the global cycling of elements and contains a large reservoir of organic carbon. This paper reports the development of a DNA extraction protocol that addresses the particular problems faced in applying molecular ecological techniques to samples containing very low biomass. Sediment samples were collected from different geographical locations within the Pacific Ocean and include the Ocean Drilling Program (ODP) Leg 190, Nankai Trough Accretionary Prism. Seven DNA extraction protocols were tested and a commercially available DNA extraction kit with modifications was shown to produce higher yields of polymerase chain reaction (PCR)-amplifiable DNA than standard laboratory methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene diversity revealed that template DNA from these extremely low biomass sediment samples was susceptible to PCR bias and random amplification. We propose that it is essential to screen 16S rRNA gene products for bacterial diversity by DGGE or other rapid fingerprinting methods, prior to their use in establishing a representative clone library of deep sub-seafloor bacteria. This represents a cautionary approach to analysis of microbial diversity in such sub-seafloor ecosystems.
对深海环境的调查表明，在全球范围内，沉积物深处存在大量的微生物生物量。现在人们认为，这个深部生物圈在全球元素循环中起着重要作用，并且含有大量的有机碳储备。本文报道了一种DNA提取方法的开发，该方法解决了将分子生态学技术应用于生物量极低的样本时所面临的特殊问题。沉积物样本采集自太平洋不同地理位置，包括大洋钻探计划（ODP）第190航次、南海海槽增生楔。测试了七种DNA提取方法，结果表明，一种经过改良的市售DNA提取试剂盒比标准实验室方法能产生更高产量的可用于聚合酶链反应（PCR）扩增的DNA。对16S rRNA基因多样性的变性梯度凝胶电泳（DGGE）分析表明，这些生物量极低的沉积物样本中的模板DNA容易受到PCR偏差和随机扩增的影响。我们建议，在用于建立深部海底细菌代表性克隆文库之前，必须通过DGGE或其他快速指纹图谱方法对16S rRNA基因产物进行细菌多样性筛选。这是分析此类海底生态系统中微生物多样性的一种谨慎方法。
