DNA Stable-Isotope Probing (DNA-SIP)

Abstract

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...

Alpha- and Gammaproteobacterial Methanotrophs Codominate the Active Methane-Oxidizing Communities in an Acidic Boreal Peat Bog

“…DNA was extracted from frozen peat samples with a MoBio PowerSoil DNA kit according to the manufacturer's protocol and stored at Ϫ20°C until further analysis. Stable isotope probing was conducted as described previously (28,29). In brief, extracted DNA was added to a cesium chloride solution and centrifuged by ultracentrifugation at 177,000 ϫ g. After 40 h, samples were removed from the ultracentrifuge and fractionated by needle fractionation into 12 or 13 fractions, and the density of each fraction was determined with a digital refractometer (Reichart AR200).…”

“…DNA was precipitated from all fractions with polyethylene glycol and glycogen as a carrier (28,29). Precipitated DNA was stored at Ϫ20°C until further analysis.…”

Diverse, uncultivated bacteria and archaea underlying the cycling of dissolved protein in the ocean

“…The protocol for density gradient centrifugation and gradient fractionation followed previously described methods for DNA-SIP (Neufeld et al, 2007;Dunford and Neufeld, 2010), with some minor modifications. In brief, density gradient centrifugation was performed in a TV90 vertical rotor at 20°C for 40 h at 177 000 g in an Optima XL-90 ultracentrifuge (Beckman Coulter, Brea, CA, USA).…”

Stable isotope probing in the metagenomics era: A bridge towards improved bioremediation

“…After stable isotopes have been pulsed into the environment and metabolically active cells have incorporated the label into their biomass, biomarkers are recovered and analyzed (Neufeld et al, 2007b, Dunford and Neufeld, 2010) (Figure 1). Therefore, SIP is an approach that can identify microbial populations with a defined function.…”