title: Extraction of high molecular weight DNA from microbial mats.
abstract: Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.
authors.affiliation: Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, USA.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Bey
authors.forename: Erin B
authors.initials: EB
authors.lastname: Fichot
authors.forename: R Sean
authors.initials: RS
authors.lastname: Norman
mesh_terms: D001419:Bacteria / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification*; D019015:Geologic Sediments / Q000382:microbiology*; D008970:Molecular Weight; D054712:Salinity; D014871:Water Microbiology
chemical_list: D004269:DNA, Bacterial