Multiple and simultaneous detection of specific bacteria in enriched bacterial communities using a DNA microarray chip with randomly generated genomic DNA probes.
A DNA microarray chip for detecting the presence of specific bacterial strains was developed using random genomic probes derived from genomic DNA, i.e., without any sequence information. Thirteen bacteria from different genuses were selected as targets. For the fabrication of the random genomic probes, genomic DNA from pure cultures of each bacterium was fractionated using several pairs of restriction endonucleases. After size fractionation of the genomic DNA fragments, random genomic libraries for each bacterium were constructed. From the library, specific probes were amplified by PCR and the probes were affixed to a slide glass to fabricate the DNA microarray chip. The results from tests with pure and mixed cultures of the bacteria used in the fabrication of the chips showed specific responses and only a small portion of cross-hybridization. This DNA microarray chip was also tested to detect the presence of specific bacteria in mixed populations. In these tests, it was demonstrated that this system provided a fast and specific response to the presence of bacterial species in mixed samples, even in activated sludge samples. This indicates that any DNA microarray chip for the detection of specific bacteria can be fabricated using the same protocols as presented in this study without requiring any genus level sequence information from pure isolates.
Kim
Byoung Chan
BC
Advanced Environmental Monitoring Research Center (ADEMRC) and National Research Laboratory on Environmental Biotechnology, Gwangju Institute of Science and Technology (GIST), 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea.
Park
Ji Hyun
JH
Gu
Man Bock
MB
D015373:Bacterial Typing Techniques / Q000379:methods*; D015342:DNA Probes / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D005828:Genetics, Population; D016680:Genome, Bacterial; D033601:Gordonia Bacterium / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
D015342:DNA Probes; D004269:DNA, Bacterial