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A procedure for separate recovery of extra- and intracellular DNA from a single marine sediment sample

  • Extracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Previous studies suggested that eDNA plays an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Previous methods for eDNA extraction were either not suitable for oligotrophic sediments or only allowed quantification but no genetic analyses. Our procedure is based on cell detachment and eDNA liberation from sediment particles by sequential washing with an alkaline sodium phosphate buffer followed by a separation of cells and eDNA. The separated eDNA is then bound onto silica particles and purified, whereas the intracellular DNA from the separated cells is extracted using a commercial kit. The method provides extra- and intracellular DNA of high purity that is suitable for downstream applications like PCR. Extracellular DNA was extracted from organic-rich shallow sediment of the Baltic Sea, glacially influenced sediment of the Barents Sea and from the oligotrophic South PacificExtracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Previous studies suggested that eDNA plays an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Previous methods for eDNA extraction were either not suitable for oligotrophic sediments or only allowed quantification but no genetic analyses. Our procedure is based on cell detachment and eDNA liberation from sediment particles by sequential washing with an alkaline sodium phosphate buffer followed by a separation of cells and eDNA. The separated eDNA is then bound onto silica particles and purified, whereas the intracellular DNA from the separated cells is extracted using a commercial kit. The method provides extra- and intracellular DNA of high purity that is suitable for downstream applications like PCR. Extracellular DNA was extracted from organic-rich shallow sediment of the Baltic Sea, glacially influenced sediment of the Barents Sea and from the oligotrophic South Pacific Gyre. The eDNA concentration in these samples varied from 23 to 626 ng g(-1) wet weight sediment. A number of experiments were performed to verify each processing step. Although extraction efficiency is higher than other published methods, it is not fully quantitative. (C) 2014 Elsevier B.V. All rights reserved.show moreshow less

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Metadaten
Author:Mashal AlawiORCiDGND, Beate Schneider, Jens KallmeyerORCiDGND
DOI:https://doi.org/10.1016/j.mimet.2014.06.009
ISSN:0167-7012 (print)
ISSN:1872-8359 (online)
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=24955890
Parent Title (English):Journal of microbiological methods
Publisher:Elsevier
Place of publication:Amsterdam
Document Type:Article
Language:English
Year of first Publication:2014
Year of Completion:2014
Release Date:2017/03/27
Tag:Ancient DNA; Extracellular DNA; Fossil DNA; Intracellular DNA; South Pacific Gyre; eDNA
Volume:104
Pagenumber:7
First Page:36
Last Page:42
Funder:Helmholtz Society and the German Science Foundation (DFG) through the ODP/IODP Priority program
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Erd- und Umweltwissenschaften
Peer Review:Referiert