Microarray-Based Analysis of Subnanogram Quantities of Microbial Community DNAs by Using Whole-Community Genome Amplification

Abstract

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r 2 ‫؍‬ 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r 2 ‫؍‬ 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r 2 ‫؍‬ 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment.Microorganisms play integral and often unique roles in ecosystem functions, yet we often know little about dominant populations that presumably perform these functions, nor do we know much about how these populations differ with habitat.

Overall functional gene diversity of microbial communities in three full-scale activated sludge bioreactors

“…All labeled DNA was resuspended in a 10-μl hybridization solution as previously described (Wu et al 2006) and was hybridized with GeoChip 4.2 on a MAUI hybridization station (BioMicro, Salt Lake City, UT, USA) at 42°C with 40 % formamide for 16 h. Microarrays were scanned by a ScanArray 5000 Microarray Analysis System (PerkinElmer, Wellesley, MA, USA) at 100 % laser power.…”

Microbial community functional structure in response to antibiotics in pharmaceutical wastewater treatment systems

“…Single-strand binding protein (267 ng/ml) and spermidine (0.1 mM) were also added to the reaction mix to improve the amplification efficiency (Wu et al, 2006). The reactions were incubated at 30 C for 10 h and stopped by heating the mixtures at 65 C for 10 min.…”

GeoChip-based analysis of the microbial community functional structures in simultaneous desulfurization and denitrification process

“…The amplified DNA was labeled using Cy5 fluorescent dye (GE Healthcare, USA) with random primer and the labeled DNA was purified by QIAquick PCR purification column (Qiagen, USA) using protocols described previously (Wu et al, 2008). The purified DNA was dried and resuspended in 130 μL hybridization solution (Wu et al, 2006). Hybridization process was carried out in a Tecan HS 4800 Pro Hybridization Station (Tecan US, USA) at 42°C for 10 hr.…”