DNA stable-isotope probing


Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4–5 days, with much of the time being committed to the ultracentrifugation step.

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Figure 1: Retrieval of DNA from ultracentrifuge gradients.
Figure 2: Expected results for SIP gradient fractionation.
Figure 3: Expected results for an EtBr-containing gradient.


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This work was supported by funding from the Natural Environment Research Council (UK), the Deutsche Forschungsgemeinschaft (SFB 395), the Max Planck Society, the Helmholtz Society and the Natural Sciences and Engineering Research Council (Canada).

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Correspondence to Josh D Neufeld or J Colin Murrell.

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The authors declare no competing financial interests.

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Neufeld, J., Vohra, J., Dumont, M. et al. DNA stable-isotope probing. Nat Protoc 2, 860–866 (2007). https://doi.org/10.1038/nprot.2007.109

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