Dunford Eric A, Neufeld Josh D
Department of Biology, University of Waterloo.
J Vis Exp. 2010 Aug 2(42):2027. doi: 10.3791/2027.
DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.
DNA稳定同位素探测（DNA-SIP）是一种强大的技术，用于识别将特定碳底物和营养物质同化到细胞生物量中的活性微生物。因此，这种不依赖培养的技术一直是一种重要的方法，用于将代谢功能赋予广泛存在于陆地和水生环境中的各种群落。在用稳定同位素标记的化合物孵育环境样品后，提取的核酸进行密度梯度超速离心，随后进行梯度分级分离，以分离不同密度的核酸。从氯化铯中纯化DNA可获取标记和未标记的DNA，用于后续的分子表征（如指纹图谱、微阵列、克隆文库、宏基因组学）。本《可视实验视频杂志》视频方案提供了密度梯度超速离心、梯度分级分离和标记DNA回收方案的可视化分步解释。该方案还包括样本SIP数据，并强调了为确保成功进行DNA-SIP分析必须考虑的重要提示和注意事项。
