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

• Published in: Microbiological research Vol. 163; no. 4; pp. 403 - 407
• Main Authors: do Nascimento, Cássio, Santos Barbosa, Rodrigo Edson, Mardegan Issa, João Paulo, Watanabe, Evandro, Yoko Ito, Izabel, Monesi, Nadia, Albuquerque Junior, Rubens Ferreira de
• Format: Journal Article
• Language: English
• Published: Germany Elsevier GmbH 01.01.2008
• Subjects: Chekerboard technique; DNA Probes - chemistry; Fluorescein; Genome, Bacterial - genetics; Genomic probes; Luminescence; Nucleic Acid Hybridization - methods; Staining and Labeling - methods; Chekerboard technique; Genomic probes; Fluorescein
• ISSN: 0944-5013; 1618-0623
• DOI: 10.1016/j.micres.2006.11.020

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.