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Related Experiment Videos

DNA stable-isotope probing.

Josh D Neufeld1, Jyotsna Vohra, Marc G Dumont

  • 1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. jneufeld@uwaterloo.ca

Nature Protocols
|April 21, 2007
PubMed
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Stable-isotope probing identifies microbes without cultivation by tracking labeled substrates like carbon-13 (¹³C) or nitrogen-15 (¹⁵N) in their biomass. This method reveals functional microbial groups involved in specific metabolic processes.

Area of Science:

  • Microbial Ecology
  • Molecular Biology
  • Environmental Science

Background:

  • Stable-isotope probing (SIP) is a cultivation-independent technique for identifying active microorganisms in environmental samples.
  • It relies on the assimilation of stable-isotope labeled substrates (e.g., ¹³C or ¹⁵N) into microbial biomass.
  • Analysis of labeled nucleic acids (DNA/RNA) provides phylogenetic and functional insights into substrate-utilizing microbes.

Purpose of the Study:

  • To provide guidelines for incubating environmental samples with stable-isotope labeled substrates.
  • To present a detailed protocol for separating labeled DNA from unlabeled community DNA.
  • To optimize the recovery of labeled DNA from cesium chloride (CsCl) density gradients.

Main Methods:

  • Incubation of environmental samples with stable-isotope labeled substrates (¹³C or ¹⁵N).

Related Experiment Videos

  • Extraction of total microbial DNA from incubated samples.
  • Separation of labeled DNA from unlabeled DNA using modified CsCl density gradient ultracentrifugation.

    • Main Results:

    • A modified protocol enhances the recovery of stable-isotope labeled DNA from CsCl gradients.
    • The separation and retrieval of labeled and unlabeled DNA fractions are achievable within 4-5 days.
    • Ultracentrifugation is the most time-consuming step in the DNA separation process.

    Conclusions:

    • Stable-isotope probing, coupled with an optimized DNA separation protocol, is an effective method for studying microbial metabolism.
    • The described protocol improves the efficiency of identifying specific substrate-utilizing microorganisms in complex environmental communities.
    • This technique advances our understanding of microbial functions without the need for cultivation.