Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities

• Published in: Environmental microbiology Vol. 5; no. 10; pp. 933 - 943
• Main Authors: Denef, Vincent J., Park, Joonhong, Rodrigues, Jorge L. M., Tsoi, Tamara V., Hashsham, Syed A., Tiedje, James M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.10.2003
• Subjects: Bacteria - genetics; Biodegradation, Environmental; Genome, Bacterial; Genomics; Oligonucleotide Array Sequence Analysis - methods; Sensitivity and Specificity; Soil Microbiology
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1046/j.1462-2920.2003.00490.x

Summary Spotted oligonucleotide microarrays potentially offer a wide scope of applications for microbial ecology, especially as they improve the flexibility of design and the specificity of detection compared to PCR product based microarrays. Sensitivity, however, was expected to be problematic, as studies with the more sensitive PCR‐based cDNA microarrays indicate that only genes from populations contributing to more than 5% of the community DNA can be detected. We evaluated several parameters to increase sensitivity and then tested applicability for bacterial functional genomics. The optimal parameters were the use of 5′‐C6‐amino‐modified 70‐mers printed on CMT‐GAPS II substrates at a 40 µM concentration combined with the use of Tyramide Signal Amplification labelling. This protocol allowed detection of single copy genes belonging to an organism contributing to 1% or more of the total community. To demonstrate its application, we detected the specific aromatic oxygenase genes in a soil community degrading polychlorinated biphenyls (PCBs). This increase in sensitivity is important if oligonucleotide microarrays are to be used for simultaneous monitoring of a range of functions performed by different microorganisms in the environment.