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Applied and Environmental Microbiology, December 2001, p. 5780-5790, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5780-5790.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

Liyou Wu,1,2 Dorothea K. Thompson,1 Guangshan Li,1 Richard A. Hurt,1 James M. Tiedje,2 and Jizhong Zhou1,2,*

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831,1 and Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 488242

Received 13 March 2001/Accepted 21 September 2001

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65°C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r2 = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

* Corresponding author. Mailing address: Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, TN 37831-6038. Phone: (865) 576-7544. Fax: (865) 576-8646. E-mail: zhouj{at}ornl.gov.

Applied and Environmental Microbiology, December 2001, p. 5780-5790, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5780-5790.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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