A liquid chromatography – mass spectrometry method to measure 13C-isotope enrichment for DNA stable-isotope probing
Abstract:DNA stable-isotope probing (DNA-SIP) is a cultivation-independent technique that makes it possible to associate metabolic function and taxonomic identity in a wide range of terrestrial and aquatic environments. In DNA-SIP, DNA is labeled via the assimilation of a labeled growth substrate that is subsequently used to identify microorganisms involved in assimilation of the substrate. However, the labeling time has to be sufficient to obtain labeled DNA but not so long such that cross-feeding of 13C-labeled metabolites from the primary consumers to nontarget species can occur. Confirmation that the DNA is isotopically labeled in DNA-SIP assays can be achieved using an isotope ratio mass spectrometer. In this study, we describe the development of a method using liquid chromatography (HPLC) coupled to a quadrupole mass spectrometer (QMS) to measure the 13C enrichment of thymine incorporated into DNA in Escherichia coli cultures fed with [13C]acetate. The method involved the hydrolysis of DNA extracted from the cultures that released the nucleotides, followed by the separation of the thymine by HPLC on a reverse-phase C8 column in isocratic elution mode and the detection and quantification of 13C-labeled thymine by QMS. To mimic a DNA-SIP assay, a DNA mixture was made using 13C-labeled E. coli DNA with DNA extracted from five bacterial species. The HPLC–MS method was able to measure the correct proportion of 13C-DNA in the mix. This method can then be used as an alternative to the use of isotope ratio mass spectrometry in DNA-SIP assays.
Keywords: 13C isotopic ratio; DNA stable-isotope probing; marquage d’ADN par isotope stable; microbial ecology; quadrupole mass spectrometry; rapport isotopique de 13C; spectromètre de masse quadripole; écologie microbienne
Document Type: Research Article
Publication date: 2012-03-17
- Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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