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

Abstract

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 (r 2 ‫؍‬ 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.

GeoChip: a comprehensive microarray for investigating biogeochemical, ecological and environmental processes

“…When PCR-amplicon-based FGAs were used, the detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA without amplification of target templates (Wu et al, 2001). Studies with a 50-mer FGA showed that the detection limit without target template amplification ranges from 5-10 ng of pure genomic DNA and 50-100 ng in a mixture of genomic DNA from different organisms (Rhee et al, 2004;Tiquia et al, 2004).…”

“…Because the arrays contain probes from the genes with known biological functions, they will be useful in linking microbial diversity to ecosystem processes and functions. Several systematic experimental evaluations indicated that FGA-based microarrays can be used as specific, sensitive and potentially quantitative tools for detecting microbial populations and functional activities in natural settings (Wu et al, 2001(Wu et al, , 2006aTaroncher-Oldenburg et al, 2003;Rhee et al, 2004;Steward et al, 2004;Tiquia et al, 2004). However, one of the greatest challenges in using FGAs for detecting functional genes and/or microorganisms in the environment is to design oligonucleotide probes specific to the target genes/microorganisms of interest because sequences of a particular functional gene are highly homologous and/or incomplete, especially sequences derived from laboratory cloning of environmental samples.…”

Introduction to Microarray‐Based Detection Methods

“…[68,97,124,152,167,199]. Furthermore, PCR products can be used as probes for the functional gene arrays [31,191]. The inappropriate labeling of a substantial fraction of the PCR products (1-5%) can lead to poorly controlled microarrays, even when originating from prestigious research centers or commercial entities [89].…”

“…pmoA, amoA, nifH, nirK, nirS). Many of these have been successfully applied on various microarray platforms [21,86,171,191]. The major limitation of these alternative marker genes is the limited organism coverage of published sequence databases.…”

“…According to the nature of the marker gene, one can further distinguish phylogenetic [30,110,145] and functional MDMs, also referred to as functional gene arrays [21,171,191].…”