DNA migration is retarded when a fragment reaches its first

melting domain, allowing separation of the mixture of PCR amplicons on the gel (10–12). DGGE of PCR-amplified 16S rDNA fragments has the potential advantage of detecting multiple species and was first used for the study of total subgingival microbial populations in 2003 (7, 8). Since then, analysis of subgingival plaque samples Epacadostat order by DGGE using several different primer pairs for amplification of the 16S rDNA regions of V3, V3-V5, and V6-V8 have been described in published articles (7, 8, 13, 14). These reports suggest that DGGE is useful for microbiological investigation of subgingival microbial populations. However, no reports have focused on which primer pairs are most suitable for analyzing subgingival bacterial communities, nor on whether the choice of the primer pairs alters the DGGE results. To address these questions, in the present study the DGGE profiles of different 16S rDNA regions of periodontal pathogens were first analyzed. The target regions (V3, V3-V5, and V6-V8) of 16S rDNA from three periodontal strains were cloned in to plasmid vector and the constructed plasmids used as templates for PCR-DGGE analysis templates which could easily be manipulated in further experiments. Briefly, three type strains, P. gingivalis

ATCC 33277, Fusobacterium nucleatum ATCC 25586, and Prevotella nigrescens ATCC 33563, were cultured APO866 cost anaerobically in brain heart infusion medium broth (Becton Dickinson, Sparks, MD, USA) supplemented with 10 μg/ml hemin and 1 μg/ml vitamin K. Chromosome DNA of these type strains was extracted using a bacterial genomic DNA extraction kit (Tiangen, Beijing, China) and used as PCR templates to amplify the 16S rDNA fragments with Ex Taq DNA polymerase (Takara, Dalian, China). The primer pairs were as follows: V3-s, 5′-CCT ACG GGA GGC AGC AG-3′ and V3-a, 5′-ATT ACC GCG PLEK2 GCT GCT GG-3′ for the V3 regions; V3-s and V3/5-a,

5′-CCG TCA ATT CTT TTR AGT-3′ for the V3-V5 regions; and V6/8-s, 5′-AAC GCG AAG AAC CTT AC-3′ and V6/8-a, 5′-CGG TGT GTA CAA GAC CC-3′ for V6-V8 regions, respectively (7, 8, 14). The theoretical primer matches of these primers with Ribosomal Database Collection Release 10 (http://rdp.cme.msu.edu/probematch/search.jsp) are: V3-s, 89.6%; V3-a, 66.1%; V3/5-a, 77.6%; V6/8-a, 58.7%; and V6/8-b, 18.4%, respectively. The PCR products were cloned into the pMD18-T vector (Takara) and the resulting plasmids sent to Invitrogen (Shanghai, China) to confirm their sequence accuracy (data not shown). The purified plasmids were used as templates for DGGE analysis. To prepare the PCR fragments for DGGE analysis, GC clamps 5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G -3′and 5′-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G-3′ were added to the forward primers V3-s and V6/8-s, respectively (7, 8).