title: Development of temporal temperature gradient electrophoresis for characterising methanogen diversity.
abstract: Temporal temperature gradient electrophoretic (TTGE) analysis of 16S rDNA sequences was optimized to monitor the methanogen population present in water and sediments of a small eutrophic lake, Priest Pot, in the English Lake district. The production of nonrepresentative TTGE profiles due to the generation of polymerase chain reaction (PCR) artifacts initially proved problematical. The use of a proofreading polymerase in the PCR was found to be essential and fully optimized protocols were established and tested to ensure confidence that the TTGE profiles truly reflected sequence diversity. TTGE analysis revealed the methanogen population to be less diverse in water than in sediment. The most genetic diversity was observed in TTGE profiles of sediment DNA isolated in winter and the least was in sediment DNA isolated in summer. DNA sequencing analysis of bands recovered from TTGE gels revealed the presence of two methanogen communities. One clustered with Methanosaeta species and the other with the Methanomicrobiales. Many sequences showed low DNA sequence similarity to known methanogens, suggesting that Priest Pot harbors previously undescribed methanogen species.
authors.affiliation: School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB, UK.
authors.forename: Julie
authors.initials: J
authors.lastname: Earl
authors.forename: Roger W
authors.initials: RW
authors.lastname: Pickup
authors.forename: Donald A
authors.initials: DA
authors.lastname: Ritchie
authors.forename: Clive
authors.initials: C
authors.lastname: Edwards
mesh_terms: D017931:DNA Primers; D019641:DNA, Archaeal / Q000032:analysis* / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004784:Environmental Monitoring; D005618:Fresh Water / Q000032:analysis / Q000382:microbiology*; D019015:Geologic Sediments / Q000032:analysis / Q000382:microbiology*; D008697:Methane / Q000032:analysis; D017018:Methanomicrobiales / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D019640:Methanosarcinales / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction; D019642:RNA, Archaeal / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012621:Seasons; D013045:Species Specificity; D013696:Temperature; D006113:United Kingdom; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D019641:DNA, Archaeal; D019642:RNA, Archaeal; D012336:RNA, Ribosomal, 16S; D011092:Polyethylene Glycols; D008697:Methane