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Applied and Environmental Microbiology, April 2004, p. 2263-2270, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2263-2270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis

William E. Holben,1* Kevin P. Feris,1 Anu Kettunen,2 and Juha H. A. Apajalahti{dagger}

Microbial Ecology Program, Division of Biological Sciences, The University of Montana, Missoula, Montana 59812,1 Enteromix Research, Danisco Innovation, 02460 Kantvik, Finland2

Received 29 July 2003/ Accepted 31 December 2003

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.

* Corresponding author. Mailing address: Microbial Ecology Program, Division of Biological Sciences, The University of Montana, Missoula, MT 59812-1006. Phone: (406) 243-6163. Fax: (406) 243-6163. E-mail: bill.holben{at}mso.umt.edu.

{dagger} Present address: Alimetrics Ltd., 07900 Helsinki, Finland.

Applied and Environmental Microbiology, April 2004, p. 2263-2270, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2263-2270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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