Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification.
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 (r2 = 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 (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 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.
Wu
Liyou
L
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6038, USA.
Liu
Xueduan
X
Schadt
Christopher W
CW
Zhou
Jizhong
J
D018533:Biomass; D004269:DNA, Bacterial / Q000032:analysis*; D017753:Ecosystem; D005618:Fresh Water / Q000382:microbiology*; D016680:Genome, Bacterial; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D014876:Water Pollution
D004269:DNA, Bacterial