DNA buoyant density shifts during N-15-DNA stable isotope probing

摘要

DNA-based stable isotope probing (SIP) is a novel. technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using N-15-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BID) resulting from N-15 incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of N-15 into DNA increased BD by 0.015 +/- 0.002 g mL(-1) for E. coli and 0.013 +/- 0.002 g mL(-1) for M. luteus. The DNA BD shift was greatly increased (0.045g mL(-1)) when dual isotope (C-13 Plus N-15) labeling was employed. Despite the limited DNA BD shift following N-15 enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil. (C) 2006 Elsevier GmbH. All rights reserved.