title: Habitat specific dsrAB targeted functional gene microarray
status: Public on Nov 26 2010
contact.city: Vienna
contact.first: Doris
contact.last: Steger
contact.state: Vienna
contact.address: Althanstrasse 14
contact.department: Department of Microbial Ecology
contact.country: Austria
contact.institute: University of Vienna
organism: uncultured sulfate-reducing bacterium
manufacture_protocol: Probes targeting dsrA or dsrB were designed using the probe tools of the ARB software package and a dsrAB ARB database containing approximately 500 sequences (>1500 nucleotides) from pure cultures and environmental studies. Based on a distance matrix tree of all previously published dsrAB clone sequences retrieved from Schlöppnerbrunnen I+II system, 146 probes with an average length of 30 bases were designed targeting different clone groups from the study site. Thermodynamic properties of the probes were calculated using the two-state hybridization server of DINAMelt (default settings: linear DNA, 37°C, 1 M Na+, 0 M Mg2+, 0.1 mM strand concentration). Probes lengths were adjusted to obtain similar theoretical free energy (deltaG) values. Final probes had a length of 27 to 34 nucleotides with an average deltaG of -41.0 ± 0.7 kcal/mol. The in silico specificity and number of weighted mismatches to non-target dsrAB sequences of each probe were determined using probeCheck. Oligonucleotides for microarray spotting were synthesized by Microsynth (Balgach, Switzerland). The 5’-end of each oligonucleotide probe carried a T-spacer consisting of 30 dTTP molecules and the 5’-terminal dTTP was aminated to allow covalent coupling of the oligonucleotides to aldehyde group-coated VSS-25 glass slides. Each probe was adjusted to a concentration of 50 pmol/µl in 50% dimethyl sulfoxide and printed on the slide by using a BioRobotics MicroGrid spotter (Genomics Solutions, Ann Arbor, MI, USA) under constant temperature (20°C) and humidity (min. 50%) conditions. Each microarray contained three and six replicate spots of each dsrA/dsrB-targeted probe and control probe, respectively. Freshly spotted DNA microarrays were incubated overnight at room temperature in a humid chamber. Slides were further treated with sodium borohydride as described previously.