Evaluation of DNA quality obtained from stored human saliva and its applicability in forensic identification in Forensic Dentistry

A saliva pode ser utilizada como fonte eficiente de DNA para tecnicas de identificacao humana, as quais sao aceitas como prova legal, sendo o parecer do profissional superlativo para a formulacao da sentenca. Esse material pode ser coletado de maneira indolor e nao-invasiva e utilizado mesmo quando armazenado em diferentes condicoes. Este trabalho objetiva avaliar a qualidade do DNA obtido de saliva humana armazenada e sua aplicabilidade da identificacao de pessoas. Foram analisadas amostras salivares de n=100 sujeitos da pesquisa, coletadas nas formas de saliva in natura e saliva coletada de swab. A saliva foi armazenada a -20ºC. Apos 7 dias, realizou-se a primeira etapa, quando o DNA foi extraido das 200 amostras de saliva utilizando-se a resina InstaGene (Bio-Rad Laboratories, Inc., Hercules, CA, USA) e, posteriormente, submetido a PCR e a eletroforese. Apos 180 dias de armazenamento da saliva, repetiu-se a mesma tecnica da primeira fase, porem em apenas 20 amostras, selecionadas aleatoriamente do total de 100 amostras de saliva coletadas por swab bucal. Os resultados da primeira etapa indicaram que o DNA foi extraido com sucesso em 96% das reacoes realizadas para as 200 amostras de saliva, fato observado tambem quando se analisou as amostras em separado, de saliva in natura (94%) e saliva advinda do swab (98%). Alem disso, nao houve diferencas estatisticamente significantes na extracao do DNA entre as duas formas de coleta de saliva utilizadas. Na segunda fase, foi possivel a deteccao do gene alvo nas 20 amostras analisadas (100%). Posteriormente, objetivando-se aprofundar a analise do DNA salivar de maneira mais proxima ao padrao exigido em um processo de identificacao, o gene SIX3-2 foi testado nas amostras e tambem foi feita a digestao do produto da PCR com a enzima de restricao MbO1 para avaliar polimorfismo do gene ADRA-2. Os resultados mostraram que a quantidade e a qualidade do DNA advindo de saliva do swab bucal, bem como as tecnicas empregadas estao adequadas a analise forense do DNA. Portanto, a saliva humana e bastante util como fonte de DNA e pode ser armazenada, em temperatura e condicoes ideais, para analise posterior.The saliva can be used as efficient DNA source for human identification techniques in which they are accepted as forensic proof, being the superlative professionals opinion for the sentence formulation. This material can be collected in a painless and noninvasive way and it is used even when stored in different conditions. This paper aims at evaluating DNA quality obtained from stored human saliva and its applicability in people identification. Saliva samples from n=100 research subjects were analyzed. They were collected in two ways: in natura and swab. The saliva was stored at the temperature of -20°C. After 7 days, the first phase was performed, when the DNA was extracted from the 200 saliva samples using the InstaGene resin (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and, subsequently, submitted to PCR and electrophoresis. After 180 days of the saliva storage, the same technique used in the first phase was repeated; however, in only 20 samples, selected at random from the total of 100 ones collected from mouth swab. The results of the first phase indicated that the DNA was successfully extracted in 96% of the reactions performed for the 200 saliva samples. This fact was also observed when the separate saliva samples in natura (94%) and swab (98%) were analyzed. In addition, there were no statistically significant differences in the extraction of the DNA between the two ways used for collecting saliva. In the second phase, the target gene detection was possible in the 20 samples analyzed (100%). Subsequently, the SIX3-2 gene was tested in the samples with the objective of deepening the salivary DNA analysis as close as the standard required in an identification process. Also, the digestion of the PCR product with the enzyme of MbO1 restriction was performed to evaluate the polymorphism of the ADRA- 2 gene. The results showed that the DNA quantity and quality from the mouth swab saliva, as well as the techniques applied are suitable for the forensic analysis of DNA. Therefore, the human saliva is very useful as DNA source and can be stored in ideal temperature and conditions for further analysis