The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method.

Cássio do Nascimento, Rodrigo Edson Santos Barbosa, João Paulo Mardegan Issa, Evandro Watanabe, Izabel Yoko Ito, Nadia Monesi, Rubens Ferreira de Albuquerque Junior

Departamento de Materiais Dentários e Prótese, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, S/N, Monte Alegre, Ribeirão Preto, São Paulo, Brazil.

Microbiological research 2008

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Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.

Citation

Cássio do Nascimento, Rodrigo Edson Santos Barbosa, João Paulo Mardegan Issa, Evandro Watanabe, Izabel Yoko Ito, Nadia Monesi, Rubens Ferreira de Albuquerque Junior. The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method. Microbiological research. 2008;163(4):403-7