Owing to their vast diversity and as-yet uncultivated status, detection, characterization and quantification of microorganisms in natural settings are very challenging, and linking microbial diversity to ecosystem processes and functions is even more difficult. Microarray-based genomic technology for detecting functional genes and processes has a great promise of overcoming such obstacles. Here, a novel comprehensive microarray, termed GeoChip, has been developed, containing 24 243 oligonucleotide (50 mer) probes and covering 410 000 genes in 4150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation. The developed GeoChip was successfully used for tracking the dynamics of metal-reducing bacteria and associated communities for an in situ bioremediation study. This is the first comprehensive microarray currently available for studying biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, global climate change, ecosystem management, and environmental cleanup and restoration. It is particularly useful for providing direct linkages of microbial genes/populations to ecosystem processes and functions.

A new generation of functional gene arrays (FGAs; GeoChip 3.0) has been developed, with ∼28 000 probes covering approximately 57 000 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles, energy metabolism, antibiotic resistance, metal resistance and organic contaminant degradation. GeoChip 3.0 also has several other distinct features, such as a common oligo reference standard (CORS) for data normalization and comparison, a software package for data management and future updating and the gyrB gene for phylogenetic analysis. Computational evaluation of probe specificity indicated that all designed probes would have a high specificity to their corresponding targets. Experimental analysis with synthesized oligonucleotides and genomic DNAs showed that only 0.0036–0.025% false-positive rates were observed, suggesting that the designed probes are highly specific under the experimental conditions examined. In addition, GeoChip 3.0 was applied to analyze soil microbial communities in a multifactor grassland ecosystem in Minnesota, USA, which showed that the structure, composition and potential activity of soil microbial communities significantly changed with the plant species diversity. As expected, GeoChip 3.0 is a high-throughput powerful tool for studying microbial community functional structure, and linking microbial communities to ecosystem processes and functioning.

To optimize oligonucleotide probe design criteria, PCR products with different similarities to probes were hybridized to a functional gene microarray designed to detect homologous genes from different organisms. In contrast to more restrictive probe designs based on a single criterion, simultaneous consideration of the percent similarity (<90%), the length of identical sequence stretches (<20 bases), and the binding free energy (>؊35 kcal mol ؊1 ) was found to be predictive of probe specificity.The application of microarrays to environmental samples presents many technical challenges not encountered in the study of model laboratory cultures (2,11,12,14,15,16,18). One of the most challenging problems results from the need to specifically and reliably distinguish between homologous genes from many different organisms that may share a high degree of sequence similarity. Apart from the stringency of the hybridization conditions, hybridization specificities may be affected by a variety of probe design factors, including the overall sequence similarity, the distribution and positions of mismatching bases (5, 6), and the amount of free energy of the DNA duplexes formed by the probe and target sequences (4,8,12). However, most probe design programs and strategies rely on only one or two of the factors mentioned above to assess probe specificity. This may be satisfactory for the design of probes for pure culture studies of global gene expression patterns, as rather divergent genes of a single organism are represented on an array. However, for microarrays targeting homologous genes from diverse microbial communities, it is necessary to design and maximize the number of oligonucleotide probes originating from a set of highly similar sequences. By simultaneous consideration of multiple probe-target characteristics, it is possible to relax each single criterion while also ensuring more accurate predictions of probe-target hybridization behavior.Microarray design and experiment. PCR products from selected genes obtained from environmental clone libraries (9, 10) were labeled with fluorescent dyes and hybridized to a functional gene microarray containing 50-mer oligonucleotides. The PCR products chosen had different overall similarities (Ͼ85%, according to base-to-base comparisons after pairwise alignment) and/or shared identical sequence stretches of Ͼ15 bases for multiple probes. The oligonucleotide microarray used in this study followed the design of Rhee et al. (11).Briefly, probes were designed from sequence information for dissimilatory sulfite reductase genes (dsrA and dsrB), nitrite reductase genes (nirS and nirK), and an ammonium monooxygenase gene (amoA), which were downloaded from the NCBI database or from our own clone libraries using a modified version of PRIMEGENS software (17). Based on global optimal alignments, segments of 50 bases which had Ͻ85% nucleotide identity to the corresponding aligned regions of any of the BLAST hit sequences were selected as potential probes, also with consideration of the predicted...