Technical considerations for the use of15N-DNA stable-isotope probing for functional microbial activity in soils

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 C-13 substrates while here we evaluate feasibility and limitations of N-15-DNA stable-isotope probing (SIP) using labelled and unlabelled pure microbial cultures or soil extracts. Our results showed that N-15-DNA probing is feasible for cultures as well as soil samples. Limitations of N-15-DNA-SIP are (a) the need for relatively large quantities of DNA to visualise bands (although molecular resolution is much higher) and (b) N-15-DNA enrichment needed to ideally be > 50 at%; however, this requirement can be lowered to approx. 40 atom% N-15 with pure cultures using a modified CsCl centrifugation method (140Kg for 69h). These advances in N-15-DNA-SIP methodology open new opportunities to trace active microbial populations utilising specific N substrates in situ. Copyright © 2005 John Wiley & Sons, Ltd.