title: High abundance of ammonia-oxidizing Archaea in coastal waters, determined using a modified DNA extraction method.
abstract: Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.
authors.affiliation: Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington 98195, USA. hurakawa@fgcu.edu
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Urakawa
authors.forename: Willm
authors.initials: W
authors.lastname: Martens-Habbena
authors.forename: David A
authors.initials: DA
authors.lastname: Stahl
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D001105:Archaea / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics / Q000302:isolation & purification*; D008967:Molecular Biology / Q000379:methods*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction / Q000379:methods; D012623:Seawater / Q000382:microbiology*; D012680:Sensitivity and Specificity
chemical_list: D019641:DNA, Archaeal; D000641:Ammonia