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Survey of Oral Microbial Diversity using PCR-based Denaturing Gradient Gel Electrophoresis

¹ Department of Basic Science and Craniofacial Biology and
³ Division of Diagnostics, Infectious Disease and Heath Promotion, New York University College of Dentistry, 345 E. 24th Street, New York, NY 10010-4086, USA; and
² Department of Pediatrics, New York University School of Medicine, New York, NY 10016, USA;

View larger version (34K): [in a new window]   Figure 1. PCR-DGGE analysis of 16S rDNA gene fragments of ATCC type strains. (A) The PCR-amplified 16S rDNA of the ATCC type-strains shows the same 350-bp amplicons for various oral bacteria on a 1% agarose gel. (B) Denaturing gradient gel electrophoresis profiles of PCR-amplified 16S rDNA of various oral bacteria arranged from low to high %GC content. The denaturing gradient ranged from 30% to 70%.

View larger version (78K): [in a new window]   Figure 2. PCR-DGGE profiles and profile analysis of 16S rDNA gene fragments of clinical samples. (A) PCR-DGGE profiles of oral bacterial 16S rDNA from the total cultivated bacteria of 20 clinical patients with different caries status. The number of detected and dominant fragments is more pronounced in Lanes 1 to 9 (caries-free individuals) than in Lanes 10 to 20 (caries-active individuals). Lane R consists of the DGGE reference markers created according to the %GC content of the 16S rDNA amplicons and their migrating behaviors. 1, F. nucleatum vincentii (ATCC49256); 2, F. nucleatum nucleatum (ATCC25586); 3, S. sanguinis (ATCC10556); 4, S. oralis (ATCC35037); 5, S. salivarius (ATCC7073); 6, S. mutans (ATCC700610); 7, L. paracasei subsp (ATCC25598); 8, P. gingivalis (ATCC33277); 9, A. odontolyticus (ATCC17929); and 10, A. naeslundii genospecies-1 (ATCC12104). (B) A standard graph generated by the Diversity Database software for comparison of the relative distributions and the intensity profiles of multiple lanes from the DGGE image. The X-axis of the graph is the retention factor (Rf) value (from 0.0 to 1.0), and the Y-axis is the pixel intensity value at each point along the lane. The graph demonstrates the differences in the distribution and the degree of intensity of the detected bands representing S. mutans between a caries-active (CA #12) and a caries-free (CF #6) individual.