Comparison of membranes and glass slides for 16S rDNA array analyses of microbial communities by sequence-specific labeling of DNA probes.
Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for a DNA array based analyzes of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions, and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Arrays prepared on positively charged nylon membranes and coated glass slides were compared. The advantage of using membranes is that chromogenic signal amplification can be used for the detection. Furthermore, the chromogenic detection does not require any sophisticated equipment. The advantage of the glass slides is that multiple fluorescence colors can be detected simultaneously, and that internal controls can be used for normalization. This approach is also suited for high throughput screenings.
Rudi
Knut
K
MATFORSK, Norwegian Food Research Institute, Osloveien, As, Norway. knut.rudi@matforsk.no
Treimo
Janneke
J
Nissen
Hilde
H
Vegarud
Gerd
G
D001419:Bacteria / Q000235:genetics*; D015342:DNA Probes / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005898:Glass; D008567:Membranes, Artificial; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
D015342:DNA Probes; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D008567:Membranes, Artificial; D012336:RNA, Ribosomal, 16S