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      A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

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          Abstract

          A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals.

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          Most cited references64

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          Rapid isolation of high molecular weight plant DNA.

          A method is presented for the rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which is free of contaminants which interfere with complete digestion by restriction endonucleases. The procedure yields total cellular DNA (i.e. nuclear, chloroplast, and mitochondrial DNA). The technique is ideal for the rapid isolation of small amounts of DNA from many different species and is also useful for large scale isolations.
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            Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean.

            Nitrification, the microbial oxidation of ammonia to nitrite and nitrate, occurs in a wide variety of environments and plays a central role in the global nitrogen cycle. Catalyzed by the enzyme ammonia monooxygenase, the ability to oxidize ammonia was previously thought to be restricted to a few groups within the beta- and gamma-Proteobacteria. However, recent metagenomic studies have revealed the existence of unique ammonia monooxygenase alpha-subunit (amoA) genes derived from uncultivated, nonextremophilic Crenarchaeota. Here, we report molecular evidence for the widespread presence of ammonia-oxidizing archaea (AOA) in marine water columns and sediments. Using PCR primers designed to specifically target archaeal amoA, we find AOA to be pervasive in areas of the ocean that are critical for the global nitrogen cycle, including the base of the euphotic zone, suboxic water columns, and estuarine and coastal sediments. Diverse and distinct AOA communities are associated with each of these habitats, with little overlap between water columns and sediments. Within marine sediments, most AOA sequences are unique to individual sampling locations, whereas a small number of sequences are evidently cosmopolitan in distribution. Considering the abundance of nonextremophilic archaea in the ocean, our results suggest that AOA may play a significant, but previously unrecognized, role in the global nitrogen cycle.
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              Archaea in coastal marine environments.

              E Delong (1992)
              Archaea (archaebacteria) are a phenotypically diverse group of microorganisms that share a common evolutionary history. There are four general phenotypic groups of archaea: the methanogens, the extreme halophiles, the sulfate-reducing archaea, and the extreme thermophiles. In the marine environment, archaeal habitats are generally limited to shallow or deep-sea anaerobic sediments (free-living and endosymbiotic methanogens), hot springs or deep-sea hydrothermal vents (methanogens, sulfate reducers, and extreme thermophiles), and highly saline land-locked seas (halophiles). This report provides evidence for the widespread occurrence of unusual archaea in oxygenated coastal surface waters of North America. Quantitative estimates indicated that up to 2% of the total ribosomal RNA extracted from coastal bacterioplankton assemblages was archaeal. Archaeal small-subunit ribosomal RNA-encoding DNAs (rDNAs) were cloned from mixed bacterioplankton populations collected at geographically distant sampling sites. Phylogenetic and nucleotide signature analyses of these cloned rDNAs revealed the presence of two lineages of archaea, each sharing the diagnostic signatures and structural features previously established for the domain Archaea. Both of these lineages were found in bacterioplankton populations collected off the east and west coasts of North America. The abundance and distribution of these archaea in oxic coastal surface waters suggests that these microorganisms represent undescribed physiological types of archaea, which reside and compete with aerobic, mesophilic eubacteria in marine coastal environments.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                19 May 2015
                2015
                : 6
                : 476
                Affiliations
                [1] 1Department of Bioscience, Center for Geomicrobiology, Aarhus University Aarhus, Denmark
                [2] 2Department of Chemistry, University of Duisburg-Essen Essen, Germany
                [3] 3Department of Land Resources and Environmental Sciences, Montana State University Bozeman, MT, USA
                [4] 4Microbiology Section, Department of Bioscience, Aarhus University Aarhus, Denmark
                [5] 5Department of Physics and Astronomy, Stellar Astrophysics Centre, Aarhus University Aarhus, Denmark
                Author notes

                Edited by: Virginia P. Edgcomb, Woods Hole Oceanographic Institution, USA

                Reviewed by: M. J. L. Coolen, Curtin University, Australia; Konstantinos Ar. Kormas, University of Thessaly, Greece

                *Correspondence: Mark A. Lever, Department of Environmental Systems Sciences, Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal Institute of Technology Zurich, Universitätsstraße 16, CHN F37.1, CH-8092 Zurich, Switzerland mark.lever@ 123456usys.ethz.ch

                This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

                †Present Address: Mark A. Lever and Philip Eickenbusch, Department of Environmental Systems Sciences, Institute of Biogeochemistry and Pollutant Dynamics, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland

                Article
                10.3389/fmicb.2015.00476
                4436928
                26042110
                c2ec916b-1e85-4a2e-bfe4-086c3a0469ab
                Copyright © 2015 Lever, Torti, Eickenbusch, Michaud, Šantl-Temkiv and Jørgensen.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 06 April 2015
                : 30 April 2015
                Page count
                Figures: 11, Tables: 4, Equations: 0, References: 79, Pages: 25, Words: 20395
                Categories
                Microbiology
                Methods

                Microbiology & Virology
                dna,rna,extraction,environmental sample,low biomass,modular,intracellular,extracellular

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