Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities
Article first published online: 11 AUG 2003
Volume 5, Issue 10, pages 933–943, October 2003
How to Cite
Denef, V. J., Park, J., Rodrigues, J. L. M., Tsoi, T. V., Hashsham, S. A. and Tiedje, J. M. (2003), Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities. Environmental Microbiology, 5: 933–943. doi: 10.1046/j.1462-2920.2003.00490.x
- Issue published online: 11 AUG 2003
- Article first published online: 11 AUG 2003
- Received 31 January, 2003; revised 19 May, 2003; accepted 21 May, 2003.
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.