Fluorescent detection of cyanobacterial DNA using bacterial magnetic particles on a MAG-microarray.
Bacterial magnetic particles (BMPs) were used for the identification of cyanobacterial DNA. Genus-specific oligonucleotide probes for the detection of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and Synechococcus spp. were designed from the variable region of the cyanobacterial 16S rDNA of 148 strains. These oligonucleotide probes were immobilized on BMPs via streptavidin-biotin conjugation and employed for magnetic-capture hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. Bacterial magnetic particles were magnetically concentrated, spotted in 100-microm-size microwell on MAG-microarray, and the fluorescent detection was performed. This work details the development of an automated technique for the magnetic isolation, the concentration of hybridized DNA, and the detection of specific target DNA on MAG-microarray. The entire process of hybridization and detection was automatically performed using a magnetic-separation robot and all five cyanobacterial genera were successfully discriminated.
Matsunaga
T
T
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. tmatsuna@cc.tuat.ac.jp
Nakayama
H
H
Okochi
M
M
Takeyama
H
H
D001483:Base Sequence; D000458:Cyanobacteria / Q000145:classification / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D008280:Magnetics; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013050:Spectrometry, Fluorescence
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S