Análise comparativa de métodos de extração de DNA genômico de células do sangue e do leite de pequenos ruminantes

Abstract

Background: Genomic DNA (gDNA) extraction allows genetic material accessible to apply molecular techniques for diagnostic and/or genomic characterization. The main goal of this work was to analyze comparatively three manual methods of genomic DNA extraction - DNAzol, FTA and Silica - evaluating quality and yield of the nucleic acids obtained. Materials, Methods & Results: Small ruminant blood and milk samples were submitted to the three extraction methods and maintained at - 20ºC for different periods of time: four to twelve months. DNA sample purity was estimated by spectrophotometric determination through the ratio between the optical density values obtained at wavelengths of 260 nm and 280 nm (A260/A280), and, indirectly, by the PCR amplification of the GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) housekeeping gene. DNA concentration was also determinate by spectrophotometry. In order to validate the comparison, statistical analysis were performed with the variables purity, concentration and PCR from the three different methods to both samples blood and milk. The cost and the time consumed to each method were estimated considering the reagents used. The results obtained have showed a great purity level (mean values of 1.61 to 1.88) in the samples extracted by DNAzol and Silica, maintained during 4 months at - 20ºC. Percentage of PCR positive results were greater to the samples extracted by DNAzol from blood as well as from milk. Although the highest DNA concentrations were detected in the samples extracted by FTA, this method has presented the lowest values of purity and the lowest percentage of PCR positive results. When comparing the two types of samples, the average of all PCR positive results was greater to the samples extracted from blood. In the same way, when analyzing the mean purity value all samples included, blood samples presented a better ratio than the milk counterparts, suggesting the presence of PCR inhibitors in the latter. Discussion: The results obtained did not permit to establish a relationship among sample time of maintenance at - 20ºC and variables of purity and concentration. On the other hand, PCR positive results were significantly greater to the samples extracted by DNAzol and FTA maintained frozen for 12 and 9 months than to those maintained for 4 months. The DNAzol extraction cost was slightly superior to the other two methods which presented similar results. The time consumed by the three methods was equally similar for both samples analyzed. Statistical analysis has permitted to confirm the results obtained, as well as show the reproducibility of the extraction methods. In the conditions used in this work, the method that presented the best performance was DNAzol followed by Silica, regardless of the sample type. Genomic DNA samples from milk have showed the lowest percentage of PCR positive results which suggest the need to include additional protocol steps in order to eliminate putative inhibitors of PCR. In conclusion, the variables analyzed have permitted determining the best extraction method to obtain gDNA to be used in PCR.