title: 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.
authors: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. jneufeld@uwaterloo.ca
authors: Josh D
authors: JD
authors: Neufeld
authors: Jyotsna
authors: J
authors: Vohra
authors: Marc G
authors: MG
authors: Dumont
authors: Tillmann
authors: T
authors: Lueders
authors: Mike
authors: M
authors: Manefield
authors: Michael W
authors: MW
authors: Friedrich
authors: J Colin
authors: JC
authors: Murrell
mesh_terms: D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D002247:Carbon Isotopes; D004269:DNA, Bacterial; D004247:DNA