Abstract
This detailed protocol describes an approach for combining DNA stable-isotope probing-based enrichment, multiple displacement amplification (MDA), and metagenomics. Together, these three methodologies enable selective access to the genomes of uncultivated organisms that actively grow using isotopically labelled carbon and nitrogen sources. Incubations with stable-isotope-labelled substrates enrich isotopically labelled DNA from functionally relevant micro-organisms; this serves as a filter to reduce the complexity of the metagenome. The MDA step generates sufficient DNA from labelled nucleic acid for metagenomic library construction. Subsequently, genome fragments can be subjected to a variety of screens for phylogenetic or functional genes relevant to active community members. The MDA-generated DNA can also serve as template for direct high-throughput sequencing to aid reconstruction of metabolic pathways of those active organisms. Recent proof-of-concept studies have demonstrated that this novel combination of molecular methods can offer substantial enhancements to gene detection frequencies and may have great future potential for the discovery of novel genes, enzymes, and metabolic pathways.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Handelsman, J. (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68, 669–685.
Rusch, D.B., Halpern, A.L., Sutton, G., Heidelberg, K.B., Williamson, S., Yooseph, S., et al. (2007) The Sorcerer II Global Ocean SampÂling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol 5, e77.
Quince, C., Curtis, T.P., and Sloan, W.T. (2008) The rational exploration of microbial diversity. ISME J 2, 997–1006.
Radajewski, S., Ineson, P., Parekh, N.R., and Murrell, J.C. (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403, 646–649.
Dumont, M.G. and Murrell, J.C. (2005) Stable isotope probing – linking microbial identity to function. Nat Rev Microbiol 3, 499–504.
Neufeld, J.D., Wagner, M., and Murrell, J.C. (2007) Who eats what, where and when? Isotope-labelling experiments are coming of age. ISME J 1, 103–110.
Friedrich, M.W. (2006) Stable-isotope probing of DNA: insights into the function of uncultivated microorganisms from isotopically labeled metagenomes. Curr Opin Biotechnol 17, 59–66.
Schloss, P.D. and Handelsman, J. (2003) Biotechnological prospects from metagenoÂmics. Curr Opin Biotechnol 14, 303–310.
Wellington, E.M., Berry, A., and Krsek, M. (2003) Resolving functional diversity in relation to microbial community structure in soil: exploiting genomics and stable isotope probing. Curr Opin Microbiol 6, 295–301.
Neufeld, J.D., Dumont, M.G., Vohra, J., and Murrell, J.C. (2007) Methodological considerations for the use of stable isotope probing in microbial ecology. Microb Ecol 53, 435–442.
Dumont, M.G., Radajewski, S.M., Miguez, C.B., McDonald, I.R., and Murrell, J.C. (2006) Identification of a complete methane monooxygenase operon from soil by combining stable isotope probing and metagenomic analysis. Environ Microbiol 8, 1240–1250.
Binga, E.K., Lasken, R.S., and Neufeld, J.D. (2008) Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology. ISME J 2, 233–241.
Chen, Y., Dumont, M.G., Neufeld, J.D., Bodrossy, L., Stralis-Pavese, N., McNamara, N.P., et al. (2008) Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands. Environ Microbiol 10, 2609–2622.
Neufeld, J.D., Chen, Y., Dumont, M.G., and Murrell, J.C. (2008) Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics. Environ Microbiol 10, 1526–1535.
Neufeld, J.D., Vohra, J., Dumont, M.G., Lueders, T., Manefield, M., Friedrich, M.W., et al. (2007) DNA stable-isotope probing. Nat Protoc 2, 860–866.
Chen, Y. and Murrell, J.C. (2008) DNA-Stable isotope probing: recent advances and protocols. In: Aquino de Muro, M. and Rapley, R. Ed. Methods in Molecular Biology – Gene Probes. Humana Press, Totowa, NJ.
Lasken, R.S. and Stockwell, T.B. (2007) Mechanism of chimera formation during the multiple displacement amplification reaction. BMC Biotechnol 7, 19.
Zhang, K., Martiny, A.C., Reppas, N.B., Barry, K.W., Malek, J., Chisholm, S.W., et al. (2006) Sequencing genomes from single cells by polymerase cloning. Nat Biotechnol 24, 680–686.
Mußmann, M., Hu, F.Z., Richter, M., de Beer, D., Preisler, A., Jorgensen, B.B., et al. (2007) Insights into the genome of large sulfur bacteria revealed by analysis of single filaments. PLoS Biol 5, e230.
Schmeisser, C., Steele, H., and Streit, W.R. (2007) Metagenomics, biotechnology with non-culturable microbes. Appl Microbiol Biotechnol 75, 955–962.
Hrvatin, S. and Piel, J. (2007) Rapid isolation of rare clones from highly complex DNA libraries by PCR analysis of liquid gel pools. J Microbiol Methods 68, 434–436.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, USA.
Hutchison, C.A. III and Venter, J.C. (2006) Single-cell genomics. Nat Biotechnol 24, 657–658.
Acknowledgements
Natural Environment Research Council (NERC), the Deutsche Forschungsgemeinschaft (DFG), and the Max Planck Society are acknowledged for financial support. JDN acknowledges funding from a Discovery Grant from the National Sciences and Engineering Research Council of Canada (NSERC).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press
About this protocol
Cite this protocol
Chen, Y., Neufeld, J.D., Dumont, M.G., Friedrich, M.W., Murrell, J.C. (2010). Metagenomic Analysis of Isotopically Enriched DNA. In: Streit, W., Daniel, R. (eds) Metagenomics. Methods in Molecular Biology, vol 668. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-823-2_4
Download citation
DOI: https://doi.org/10.1007/978-1-60761-823-2_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-822-5
Online ISBN: 978-1-60761-823-2
eBook Packages: Springer Protocols