DNA stable-isotope probing

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Amann, R.I., Ludwig, W. & Schleifer, K.-H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Radajewski, S., Ineson, P., Parekh, N. & Murrell, J. Stable-isotope probing as a tool in microbial ecology. Nature 403, 646–649 (2000).

    CAS  Article  Google Scholar 

  3. 3

    Dumont, M.G. & Murrell, J.C. Stable isotope probing-linking microbial identity to function. Nat. Rev. Microbiol. 3, 499–504 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Neufeld, J.D., Dumont, M.G., Vohra, J. & Murrell, J.C. Methodological considerations for the use of stable isotope probing in microbial ecology. Microb. Ecol. (doi: 10.1007/s00248-006-9125-x).

  5. 5

    Evershed, R.P. et al. 13C-labelling of lipids to investigate microbial communities in the environment. Curr. Opin. Biotechnol. 17, 72–82 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Friedrich, M.W. Stable-isotope probing of DNA: insights into the function of uncultivated microorganisms from isotopically labeled metagenomes. Curr. Opin. Biotechnol. 17, 59–66 (2006).

    CAS  Article  Google Scholar 

  7. 7

    Madsen, E.L. The use of stable isotope probing techniques in bioreactor and field studies on bioremediation. Curr. Opin. Biotechnol. 17, 92–97 (2006).

    CAS  Article  Google Scholar 

  8. 8

    Prosser, J.I., Rangel-Castro, J.I. & Killham, K. Studying plant–microbe interactions using stable isotope technologies. Curr. Opin. Biotechnol. 17, 98–102 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Wagner, M., Nielsen, P.H., Loy, A., Nielsen, J.L. & Daims, H. Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. Curr. Opin. Biotechnol. 17, 83–91 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Whiteley, A.S., Manefield, M. & Lueders, T. Unlocking the “microbial black box” using RNA-based stable isotope probing technologies. Curr. Opin. Biotechnol. 17, 67–71 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Whitely, A.S., Thomson, B., Leuders, T. & Manefield, M. RNA stable-isotope probing. Nat. Protocols (in the press). DOI: 10.1038/nprot.2007.115.

  12. 12

    Dumont, M.G., Radajewski, S.M., Miguez, C.B., McDonald, I.R. and Murrell, J.C. Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis. Environ. Microbiol. 8, 1240–1250 (2006).

    CAS  Article  Google Scholar 

  13. 13

    Manefield, M., Whiteley, A.S., Griffiths, R.I. & Bailey, M.J. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68, 5367–5373 (2002).

    CAS  Article  Google Scholar 

  14. 14

    Lueders, T., Manefield, M. & Friedrich, M.W. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ. Microbiol. 6, 73–78 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Cadisch, G. et al. Technical considerations for the use of 15N-DNA stable-isotope probing for functional microbial activity in soils. Rapid Commun. Mass Spectrom. 19, 1424–1428 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Cupples, A.M., Shaffer, E.A., Chee-Sanford, J.C. & Sims, G.K. DNA buoyant density shifts during 15N-DNA stable isotope probing. Microbiol. Res. (doi: 10.1016/j.micres.2006.01.016).

  17. 17

    Sambrook, J. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

    Google Scholar 

  18. 18

    Rickwood, D. (ed.) Centrifugation: A Practical Approach (IRL, Oxford, 1983).

    Google Scholar 

Download references

Acknowledgements

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).

Author information

Affiliations

Authors

Corresponding authors

Correspondence to Josh D Neufeld or J Colin Murrell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing