title: DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols.
abstract: Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1×10(6) cells/cm(3). This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.
authors: CNRS, Institut Universitaire Européen de la Mer (IUEM)-UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes (LMEE), Plouzané, France. Karine.Alain@univ-brest.fr
authors: Karine
authors: K
authors: Alain
authors: Nolwenn
authors: N
authors: Callac
authors: Maria-Cristina
authors: MC
authors: Ciobanu
authors: Yann
authors: Y
authors: Reynaud
authors: Frédérique
authors: F
authors: Duthoit
authors: Mohamed
authors: M
authors: Jebbar
mesh_terms: D044822:Biodiversity; D003080:Cold Temperature; D019641:DNA, Archaeal / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D019015:Geologic Sediments / Q000382:microbiology*; D059625:Liquid-Liquid Extraction / Q000379:methods; D056186:Metagenomics / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D019641:DNA, Archaeal; D004269:DNA, Bacterial