RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2005; 19: 1424–1428 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1908 Technical considerations for the use of 15 N-DNA stable-isotope probing for functional microbial activity in soils { Georg Cadisch 1,2 *, Mingrelia Espana 1,3 , Rachel Causey 4 , Michael Richter 2 , Eve Shaw 4 , J. Alun W. Morgan 4 , Clive Rahn 4 and Gary D. Bending 4 1 Institute of Plant Production and Agroecology in the Tropics and Subtropics (380a), University of Hohenheim, 70593 Stuttgart, Germany 2 Department of Agricultural Sciences, Imperial College London, Wye Campus, Wye TN25 5AH, UK 3 National Institute of Agricultural Research—CENIAP, A.P. 4648, Maracay, Venezuela 4 Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK Received 1 November 2004; Revised 6 March 2005; Accepted 6 March 2005 Stable-isotope DNA probing is a culture-independent technique that may provide a link between function and phylogeny of active microorganisms. The technique has been used in association with 13 C substrates while here we evaluate feasibility and limitations of 15 N-DNA stable-isotope prob- ing (SIP) using labelled and unlabelled pure microbial cultures or soil extracts. Our results showed that 15 N-DNA probing is feasible for cultures as well as soil samples. Limitations of 15 N-DNA-SIP are (a) the need for relatively large quantities of DNA to visualise bands (although molecular reso- lution is much higher) and (b) 15 N-DNA enrichment needed to ideally be >50 at%; however, this requirement can be lowered to approx. 40 atom% 15 N with pure cultures using a modified CsCl centrifugation method (140K g for 69 h). These advances in 15 N-DNA-SIP methodology open new opportunities to trace active microbial populations utilising specific N substrates in situ. Copyright # 2005 John Wiley & Sons, Ltd. Advances in molecular techniques provide increasingly more sophisticated ways to characterise the diversity of total active microbial polulations in soil or other environments. Combining stable isotopes ( 13 C, 15 N) with these advanced molecular techniques provides new approaches to achieve a better understanding of the processes and organisms that drive C and N cycling in soil-plant systems. 1 Stable-isotope probing (SIP) is a culture-independent technique that enables taxonomic identity to be linked with function in the environ- ment by providing a highly enriched microbial substrate leading to isotopically labelled microbial DNA which can be separated on density gradients. 2 The isolated isotopically enriched DNA can then be used as a template for polymerase chain reaction (PCR) using general microbial primers or spe- cific primers for functional groups (e.g. diazotroph group, proteobacteria, cyanobacteria). Amplification products can then be used for direct cloning and sequencing for phyloge- netic identification and position analysis or community pro- filing analyses (T-RFLP, DGGE) for treatment comparisons. Alternatively, assessing isotope assimilation into RNA may be a more sensitive approach since it is not dependent on cell replication while copy number in cells is higher than for DNA and incorporation of stable isotopes into RNA is fas- ter than for DNA. 3 While most work on SIP to date has been performed using enriched 13 C substrates, 3–5 no applications have so far shown its applicability for 15 N-labelled substrates. Here we assess some of the technical issues related to the application of 15 N- DNA-SIP. In particular we investigated (a) the amount of DNA required for its visualisation in CsCl isopycnic centrifugation gradients, (b) isotopic enrichment required to obtain a clear separation of bands, and (c) testing some modifications to increase the sensitivity of the centrifugation CsCl method. EXPERIMENTAL Determination of quantities of DNA required for visualisation Azospirillum lipoferum CRT1 was grown in a mineral salts medium (Azospirillum-medium) 221, DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germay 6 ). Cultures were grown on an orbital shaker at 100 rpm for 20 h at 308C. DNA was extracted in subsamples containing approx. 1  10 9 cells using the DNeasy 1 DNA- purification kit (Qiagen). Quantification of DNA concentra- tion was performed using a NanoDrop ND-1000 spectro- photometer. DNA extracts were mixed with 0.5  TE buffer to obtain the following amounts of DNA in 4.5 mL Tris- 0.5 mM EDTA (TE): 53, 26, 13, 3 and 2 mg; thereafter, CsCl was added at 1 g mL 1 solution followed by 200 mL ethidium bromide solution (10 mg mL 1 ). CsCl density gradient centri- fugation was performed using a Sorvall centrifuge with rotor Copyright # 2005 John Wiley & Sons, Ltd. *Correspondence to: G. Cadisch, Institute of Plant Production and Agroecology in the Tropics and Subtropics (380a), University of Hohenheim, 70593 Stuttgart, Germany. E-mail: cadisch@uni-hohenheim.de { Presented at the Joint European Stable Isotope Users Group Meeting, Vienna, 30 August–3 September, 2004.