Development of a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity.
We constructed a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity to develop a new technology for simultaneous detection of the functional gene diversity in environmental samples. The diversity of the monooxygenase genes associated with benzene degradation was characterized. A new polymerase chain reaction (PCR) primer set was designed using conserved regions of benzene monooxygenase gene (BO12 primer) and used for PCR-clone library analysis along with a previously designed RDEG primer which targeted the different types of benzene monooxygenase gene. We obtained 20 types of amino acid sequences with the BO12 primer and 40 with the RDEG primer. Phylogenetic analysis of the sequences obtained suggested the large diversity of the benzene monooxygenase genes. A total of 87 60-mer probes specific for each operational taxonomical unit were designed and spotted on a microarray. When genomic DNAs of single strains were used in microarray hybridization assays, corresponding sequences were successfully detected by the microarray without any false-negative signals. Hybridization with soil DNA samples showed that the microarray was able to detect sequences that were not detected in clone libraries. Constructed microarray can be a useful tool for characterizing monooxygenase gene diversity in benzene degradation.
Iwai
Shoko
S
Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan. s_iwai@env.t.u-tokyo.ac.jp
Kurisu
Futoshi
F
Urakawa
Hidetoshi
H
Yagi
Osami
O
Furumai
Hiroaki
H
D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001554:Benzene / Q000378:metabolism*; D001673:Biodegradation, Environmental; D044822:Biodiversity; D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D012988:Soil Microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D006899:Mixed Function Oxygenases; D001554:Benzene