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Applied and Environmental Microbiology, August 2009, p. 5428-5433, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00120-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil{triangledown}

Larry M. Feinstein,1 Woo Jun Sul,2 and Christopher B. Blackwood1*

Department of Biological Sciences, Kent State University, Kent, Ohio 44242,1 Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 488242

Received 18 January 2009/ Accepted 19 June 2009

DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such as Acidobacteria, Gemmatimonades, and Verrucomicrobia were higher in the first extraction than in the sixth but that the reverse was true for Proteobacteria and Actinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.

* Corresponding author. Mailing address: Department of Biological Sciences, Kent State University, Kent, OH 44242. Phone: (330) 672-3895. Fax: (330) 672-3713. E-mail: cblackwo{at}kent.edu

{triangledown} Published ahead of print on 26 June 2009.

Applied and Environmental Microbiology, August 2009, p. 5428-5433, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00120-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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