title: Primers containing universal bases reduce multiple amoA gene specific DGGE band patterns when analysing the diversity of beta-ammonia oxidizers in the environment.
abstract: The gene encoding the active site of the ammonia monooxygenase (amoA) has been exploited as molecular marker for studying ammonia-oxidizing bacteria (AOB) diversity in the environment. Primers amplifying functional genes are often degenerated and therefore produce multiple band patterns, when analysed with the Denaturing gradient gel electrophoresis (DGGE) approach. To improve the DGGE band patterns we have designed new primer sets which contain inosine residues and are specific for the amoA gene. Primers were evaluated analysing pure AOB cultures and two habitats (wastewater treatment plant, soda pools). We found that the application of inosine primers helped to reduce the apparent complexity of the DGGE band pattern. Comparison of sequences from environmental samples using either degenerated or inosine containing amoA primers retrieved both identical and additional sequences. Both primer sets seem to be limited in their ability to detect the presence of all AOB by DGGE analyses.
authors.affiliation: Institute for Chemical Engineering, Working Group Gene Technology, Vienna University of Technology, Getreidemarkt 9/166/5, Austria. rhornek@iwag.tuwien.ac.at
authors.forename: Romana
authors.initials: R
authors.lastname: Hornek
authors.forename: Andreas
authors.initials: A
authors.lastname: Pommerening-Röser
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Koops
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Farnleitner
authors.forename: Norbert
authors.initials: N
authors.lastname: Kreuzinger
authors.forename: Alexander
authors.initials: A
authors.lastname: Kirschner
authors.forename: Robert L
authors.initials: RL
authors.lastname: Mach
mesh_terms: D001317:Austria; D017931:DNA Primers / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007288:Inosine / Q000737:chemistry; D009606:Nitrosomonas / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D007288:Inosine; D010088:Oxidoreductases; C045582:ammonia monooxygenase