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

• Published in: Journal of applied oral science 2012-08, Vol.20 (4), p.467-471
• Main Authors: Küchler, Erika Calvano, Tannure, Patricia Nivoloni, Falagan-Lotsch, Priscila, Lopes, Taliria Silva, Granjeiro, Jose Mauro, Amorim, Lidia Maria Fonte
• Format: Article
• Language: English
• Subjects: Adult; Analysis of Variance; Dentistry; DENTISTRY, ORAL SURGERY & MEDICINE; DNA; DNA - isolation & purification; Electrophoresis; Female; Genetic polymorphism; Genotyping Techniques - methods; Humans; Male; Middle Aged; Mouth - cytology; Original; PCR; Polymorphism, Restriction Fragment Length - genetics; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Saliva; Spectrophotometry; Time Factors
• ISSN: 1678-7757; 1678-7765; 1678-7765; 1678-7757
• DOI: 10.1590/s1678-77572012000400013

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. 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. 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. 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.