The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method.
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.
do Nascimento
Cássio
C
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.
Santos Barbosa
Rodrigo Edson
RE
Mardegan Issa
João Paulo
JP
Watanabe
Evandro
E
Yoko Ito
Izabel
I
Monesi
Nadia
N
Albuquerque Junior
Rubens Ferreira de
RF
D015342:DNA Probes / Q000737:chemistry; D019793:Fluorescein; D016680:Genome, Bacterial / Q000235:genetics; D049449:Luminescence; D009693:Nucleic Acid Hybridization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D015342:DNA Probes; D019793:Fluorescein