Marine Sciences Research Center, State University of New York, Stony Brook, New York 11794, and Department of Microbiology, University of Umeå, S-901 87 Umeå, Sweden.
Appl Environ Microbiol. 1988 Jun;54(6):1426-9. doi: 10.1128/aem.54.6.1426-1429.1988.
We developed a simple technique for the high-yield extraction of purified DNA from mixed populations of natural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecular biological approaches to the study of microbial communities in nature. The microorganisms from near-shore marine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-mum-pore-size fluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNA was extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100 degrees C for 1.5 to 2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNA was purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresis showed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA was sufficiently pure to allow complete digestion by the restriction endonuclease Sau3AI and ligation to vector DNA. In a sample in which the extracted DNA was quantified by binding to the dye Hoechst H33258, DNA was quantitatively extracted, and 45% of the initially extracted DNA was recovered after purification. Final yields were a few micrograms of DNA per liter of seawater and were roughly 25 to 50% of the total bacterial DNA in the sample. Alternatives to the initial harvest by filtration method, including continuous-flow centrifugation and thin-channel or hollow-fiber concentration followed by centrifugation, were less efficient than filtration in terms of both time and yield, largely because of the difficulty of centrifuging the very small bacteria typical of marine plankton. These methods were judged to be less appropriate for studies of natural populations as they impose a strong selection for the larger bacteria.
我们开发了一种从自然浮游海洋微生物（主要是细菌）混合种群中高效提取纯化 DNA 的简单技术。这是对自然环境中微生物群落进行几种分子生物学研究的必要步骤。从近海海水和半咸水样本中采集体积为 8 至 40 升的微生物，在通过玻璃纤维过滤器预过滤后，用 0.22 微米孔径的氟碳基过滤器收集，以去除大部分真核生物。直接从过滤器中提取 DNA，将过滤器浸泡在加热至 95 至 100 摄氏度的 1%十二烷基硫酸钠中 1.5 至 2 分钟。该程序基本上可以裂解所有细菌，并且不会使 DNA 明显变性。通过苯酚抽提纯化 DNA，并采取措施尽量减少剪切。琼脂糖凝胶电泳显示，最终制备物的大部分具有较大的分子大小（>23 千碱基对）。DNA 足够纯净，可以完全被限制性内切酶 Sau3AI 消化，并与载体 DNA 连接。在一个通过与染料 Hoechst H33258 结合定量提取提取 DNA 的样本中，DNA 被定量提取，并且在纯化后回收了初始提取的 DNA 的 45%。最终产量为每升海水几微克 DNA，大致占样品中总细菌 DNA 的 25%至 50%。与初始过滤方法相比，包括连续流离心和薄通道或中空纤维浓缩后离心的替代方法在时间和产量方面效率都较低，这主要是因为很难离心典型的海洋浮游生物的非常小的细菌。这些方法被认为不太适合自然种群的研究，因为它们对较大的细菌有很强的选择性。
