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Technical considerations for the use of 15 N‐DNA stable‐isotope probing for functional microbial activity in soils
Author(s) -
Cadisch Georg,
Espana Mingrelia,
Causey Rachel,
Richter Michael,
Shaw Eve,
Morgan J. Alun W.,
Rahn Clive,
Bending Gary D.
Publication year - 2005
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1908
Subject(s) - stable isotope probing , chemistry , dna , isotope , centrifugation , stable isotope ratio , soil water , microorganism , chromatography , environmental chemistry , computational biology , nanotechnology , biochemistry , bacteria , genetics , biology , ecology , physics , quantum mechanics , materials science
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 probing (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 resolution 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.