Extração do DNA genomico a partir de celulas epiteliais bucais utilizando acetato de amonio / DNA purification from buccal epithelial cells by ammonium acetate method




Buccal cells provide a convenient source of DNA for diagnosis and epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in the mouthwash solution over time. Finally, we analysed if the methodology could be used for PCR amplification of high molecular mass fragments. The procedures used in this paper avoid the use of any organic solvent. This is achieved by salting out the cellular proteins by dehydration and precipitation with a 8 M ammonium acetate solution. A group of 65 consenting subjects undertook a mouthwash containing 5mL of 3% dextrose solution. Three mL of TNE solution [17 mM Tris-HCl (pH 8.0), 50 mM NaCl and 7 mM EDTA] diluted in 66% ethanol, was added to the tube. The mouthwash solution of 20 individuals was divided into 4 tubes. One tube was used for immediate extraction (equally to the others 45 samples) and the three remaining were kept at room temperature for periods of 2, 15 and 30 days, followed by DNA isolation. Buccal cells were pelleted and resuspended in a solution containing 10 mM Tris (pH 8.0), 0.5% SDS, 5 mM EDTA, and proteinase K (20 mg/mL). After overnight incubation at 55°C proteins were removed by adding 8 M ammonium acetate followed by centrifugation. DNA was precipitated with isopropanol and resuspended in 10 mM Tris (pH 7.8) and 1 mM EDTA. PCR amplifications were performed. Three pairs of primers were used: PAX9 (202 pb); KLK (555 pb); MMP20 (1434 pb). Our analyses provided consistent evidences that DNA yield extracted by this methodology is sufficient for several PCR amplifications. Large size products (1434 bp) could be successfully obtained. Additionally, dilution of the mouthwashes in TNE/ethanol prevented DNA degradation at room temperature for periods of up to 30 days, allowing safe storage and transport of field specimens collected in isolated communities, distant from the laboratory. On conclusion, the protocol described here is quick, simple to perform, sensitive, economical and several samples can be processed at the same time


dna mouth mucosa reação em cadeia da polimerase polymerase chain reaction mucosa bucal

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