DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis. Protocol Preparation of ReagentsDNA-SIP requires the use of reagents that should be prepared in advance of the actual procedure. The directions for preparing each reagent are listed in this section and are modified from a previous SIP protocol 1 . Sample Incubation and DNA ExtractionFor DNA-SIP incubations, samples are typically incubated with heavy-isotope carbon ( 13 C) substrate. Incubation periods and conditions (e.g. nutrient supplementation, moisture, light) will vary depending on the type of sample that is incubated and the nature of the substrate. DNA-SIP experiments have been successfully performed using a variety of single carbon compounds 2,3 , multi-carbon compounds 4,5,6 , and using labelled nitrogen 7,8 or oxygen 9 . However, a drawback to using 15 N-or 18 O-labelled compounds is the decreased physical separation of labelled nucleic acid, primarily due to the presence of fewer nitrogen and oxygen atoms in DNA and RNA relative to carbon atoms.A critical control for DNA-SIP experiments is an identical incubation established with native (e.g. 12 C) substrate. This incubation provides a subsequent comparison to ensure that any apparent labelling of nucleic acid was not an artifact of the ultracentrifugation or G+C content density differences in DNA contributing to separation 10 . It is also important to keep frozen sample material for comparison to 'light' and 'heavy' DNA, and worth including a no-substrate control to assess background population changes throughout the SIP incubation. (ddH2O) to a final volume of 500 mL. Be careful not to exceed 500 mL! Warming the solution slightly while stirring will help dissolve all of the CsCl. Aliquot the final solution in sealed aliquots. In our lab, a common storage practice is to prepare 100-mL aliquots in 125-mL serum vials, which are then crimp-sealed with butyl rubber stoppers. The sealed aliquots can be store...
Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.
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