title: GC fractionation enhances microbial community diversity assessment and detection of minority populations of bacteria by denaturing gradient gel electrophoresis.
abstract: Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fractionation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis approaches to enhance assessment of microbial community diversity and detection of minority populations of microbes. This approach employs GC fractionation as an initial step to reduce the complexity of the community in each fraction. This reduced complexity facilitates subsequent detection of diversity in individual fractions. DGGE analysis of individual fractions revealed bands that were undetected or only poorly represented when total bacterial community DNA was analyzed. Also, directed cloning and sequencing of individual bands from DGGE lanes corresponding to individual G+C fractions allowed detection of numerous phylotypes that were not recovered using a traditional random cloning and sequencing approach.
authors.affiliation: Microbial Ecology Program, Division of Biological Sciences, The University of Montana, Missoula, Montana 59812, USA. bill.holben@mso.umt.edu
authors.forename: William E
authors.initials: WE
authors.lastname: Holben
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Feris
authors.forename: Anu
authors.initials: A
authors.lastname: Kettunen
authors.forename: Juha H A
authors.initials: JH
authors.lastname: Apajalahti
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D002645:Chickens; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004463:Ecology / Q000379:methods; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny
chemical_list: D004269:DNA, Bacterial