Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR

OBJECTIVE: The aim of this study was to evaluate, by PCR-RFLP and real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. MATERIAL AND METHODS: A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by dna extraction. The concentration, purity and integrity of the dna were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 a/g (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using real-time PCR. RESULTS: There was no significant difference of dna yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and real time pcr reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by real-time pcr presented c allele for irf6 gene polymorphism (homozygous: cc; heterozygous: Ct) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. CONCLUSION: We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and pcr amplified after storage in mouthwash solution at room temperature.

DNA genotyping of oral epithelial cells by laser capture microdissection / 法医学杂志

OBJECTIVE@#The STR genotypping of trace oral epithelial cells which are microdissected by laser capture microdissection system (LCM) is explored.@*METHODS@#The oral epithelial cells are microdissected using a low-power infrared laser by VERITAS Microdissection Instrument. STR loci of Profiler Plus are detected by multiplex PCR procesures.@*RESULTS@#DNA genotyping of 7-8 oral epithelial cells are succeeded, and DNA genotyping of 3-4 oral epithelial cells are failed.@*CONCLUSION@#It is viable in genotyping of trace oral epithelial cells by Laser Capture Microdissection as a new technology of seperating single cell.

Heteroplasmy in human mtDNA control region / 法医学杂志

OBJECTIVE@#To observe the length heteroplasmy and point heteroplasmy in human mtDNA control region.@*METHODS@#The peripheral blood, buccal cell, and single hair shaft from 50 individuals and 16 family members, related in their maternallineage were analyzed by direct sequencing, and clones from 20 individuals whose mtDNA sequences have a T-C transition at 16189 nt were sequenced.@*RESULTS@#No point heteroplasmy were observed in peripheral blood, buccal cell, single hair shaft from the same individual, neither in maternally related individuals. Length heteroplasmy was observed in those individuals with a homopolymeric tract and the different clones from the same individual has different proportions of length variants, but the hair shafts from the same individual were very similar to the measurements made from blood DNA. No length heteroplasmy was observed between different tissues from the same individual.@*CONCLUSION@#mtDNA sequences have a characteristic of high consistency and genetic stability, mtDNA sequencing is a suitable tool for forensic applications such as individual identification.

Aderência de Actinobacillus actinomycetemcomitans às células epiteliais bucais: estabilidade e aspectos ultra-estruturais / Actinobacillus actinomycetemcomitans attached to oral epithelial cells: stability and ultrastructural aspects

Actinobacillus actinomycetemcomitans é considerado um importante patógeno periodontal, particularmente na periodontite juvenil localizada. O mecanismo de adesäo bacteriana às células epiteliais bucais (CEB), aos dentes e a outras bactérias, constitui-se o passo inicial na colonizaçäo e patogênese nos quadros de gengivite e periodontite. neste estudo, avaliou-se a aderência às CEB, a sua variabilidade e os aspectos ultra-estruturais de 21 isolados e de uma cepa de referência de A. actinomycetemcomitans, quando submetidos a repiques sucessivos. Todos os isolados testados aderiram às CEB e os repiques sucessivos determinaram variaçöes nas taxas de aderência de cada isolado. Os isolados que apresentaram altos índices de aderência também produziram quantidades elevadas de componentes extracelulares, tais como fímbrias, vesículas e/ou material amorfo extracelular