Avaliação da qualidade do DNA extraído de saliva humana armazenada e sua aplicabilidade na identificação em Odontologia Legal

PURPOSE: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. METHODS: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at -20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. RESULTS: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. CONCLUSION: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.OBJETIVO: Este trabalho objetivou avaliar a qualidade do DNA obtido de saliva humana armazenada e sua aplicabilidade da identificação de pessoas. METODOLOGIA: Analisaram-se amostras salivares de n=20 sujeitos da pesquisa, coletadas nas formas de saliva in natura e de swab bucal, sendo armazenadas a 20ºC. Após 7 dias, o DNA foi extraído das 40 amostras de saliva e submetido à PCR e à eletroforese. Após 180 dias repetiu-se a técnica nas 20 amostras de swab. RESULTADOS: Os resultados da primeira etapa indicaram que o DNA foi extraído com sucesso em 97,5% das reações, e, analisando-se separadamente, em 95% de saliva in natura e em 100% da saliva do swab, não havendo diferenças estatisticamente significantes entre as duas formas de saliva. Na segunda fase, o resultado foi positivo para as 20 amostras analisadas (100%). Posteriormente, para analisar a qualidade do DNA obtido da saliva humana, o gene SIX3-2 foi testado nas 20 amostras de swab bucal e foi feita a digestão do produto da PCR com a enzima de restrição MbO1 para avaliar polimorfismo do gene ADRA-2 obtendo-se resultados positivos para a maioria das amostras. CONCLUSÃO: Concluiu-se que a quantidade e a qualidade do DNA advindo de saliva e as técnicas empregadas estão adequadas à análise forense do DNA

Validation of a physical anthropology methodology using mandibles for gender estimation in a Brazilian population

Validation studies of physical anthropology methods in the different population groups are extremely important, especially in cases in which the population variations may cause problems in the identification of a native individual by the application of norms developed for different communities. OBJECTIVE: This study aimed to estimate the gender of skeletons by application of the method of Oliveira, et al. (1995), previously used in a population sample from Northeast Brazil. MATERIAL AND METHODS: The accuracy of this method was assessed for a population from Southeast Brazil and validated by statistical tests. The method used two mandibular measurements, namely the bigonial distance and the mandibular ramus height. The sample was composed of 66 skulls and the method was applied by two examiners. The results were statistically analyzed by the paired t test, logistic discriminant analysis and logistic regression. RESULTS: The results demonstrated that the application of the method of Oliveira, et al. (1995) in this population achieved very different outcomes between genders, with 100% for females and only 11% for males, which may be explained by ethnic differences. However, statistical adjustment of measurement data for the population analyzed allowed accuracy of 76.47% for males and 78.13% for females, with the creation of a new discriminant formula. CONCLUSION: It was concluded that methods involving physical anthropology present high rate of accuracy for human identification, easy application, low cost and simplicity; however, the methodologies must be validated for the different populations due to differences in ethnic patterns, which are directly related to the phenotypic aspects. In this specific case, the method of Oliveira, et al. (1995) presented good accuracy and may be used for gender estimation in Brazil in two geographic regions, namely Northeast and Southeast; however, for other regions of the country (North, Central West and South), previous methodological adjustment is recommended as demonstrated in this study

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

Estimative study of the sex in skulls from the region of Guarulhos-SP using physical antropology and DNA

A investigação do sexo é uma das análises mais importantes na identificação humana. Este trabalho teve como objetivo a determinação do sexo em crânios humanos utilizando três métodos de Antropologica Física, duas quantitativas (Forensic Data Anthropolgy Bank, FDB, 1986 e Oliveira, 1995) e uma qualitativa, (Walker, 2008), e a análise genética pela amelogenina. A amostra foi composta de 66 crânios (34 homens e 32 mulheres) do Centro de Estudo e Pesquisa em Ciências Forenses, Guarulhos, SP. As metodologias foram aplicadas por duas pesquisadoras, que desconheciam o sexo dos crânios. Para o estudo estatístico realizaram-se análise descritiva, média, desvio padrão, análise discriminante linear e logística e regressão logística. A metodologia quantivativa apresentou um acerto de 89,52%. O Método FBD teve uma acurácia de 92,31%, com a elaboração de uma fórmula utilizando as medidas Largura Bizigomática, Altura Nasal, as quais apresentaram o maior dimorfismo entre os sexos, e Altura Básio-bregma e Máximo Comprimento do Crânio. A metodologia de Oliveira et al. (1995) necessitou de ajuste para a população estudada (nova fórmula com acurácia de 76,47% em homens e 78,13% em mulheres). Para o DNA, foi possível determinar o sexo em 86,15% da amostra. Pode-se afirmar que as diferentes metodologias comportaram-se de modo semelhante e com alta acurácia para determinação do sexo. A antropologia física apresenta as vantagens de facilidade de aplicação, reprodutibilidade e baixo custo, porém, necessita de ajustes populacionais. O DNA é mais complexo, necessita de infraestrutura e insumos específicos e pode ter interferência da condição ambiental, fatores que dificultam as análises, entretanto, não precisa ser ajustado á população.The investigation of the sex is one of the most important analyzes in the human identification. This study aimed to determine the sex in human skulls using three methodologies of Physical Anthropology, two quantitative (Forensic Data Anthropology Bank, FDB, 1986 e Oliveira, 1995) and one qualitative (Walker, 2008) and genetic analysis by amelogenin. The sample was composed by 66 skulls (34 men and 32 women) from the Center for Study and Research in Forensic Science, Guarulhos, SP. The methodologies were applied by two researchers who were unaware of the craniums sexes. For the statistical analysis, there were performed descriptive analysis, average, standard deviation, linear discriminant analysis and logistic and logistic regression. The quantitative methodology presented an accuracy of 89.52%. The FBD method had an accuracy of 92.31%, with the development of a mathematical model using the measures Bizygomatic breadth, Nasal heigh, which showed the biggest dimorphism between the sexes, and Basion-bregma height and Maximum Cranial Length. The Oliveiras et al. (1995) methodology required adjustment for the studied population (new formula with an accuracy of 76.47% in men and 78.13% in women). For the DNA, it was possible to determine the sex in 86.15% of the sample. The different methodologies behaved similarly and with high accuracy in sex determination. Physical anthropology has the advantages of being easy to use, reliability and low cost, but needs population adjustments. The DNA is more complex, requires specific reagents and structure and may have interference from environmental condition, however, does not need to be adjusted to the population

Forensic tanatology: biological and legal aspects

The forensic tanatology is a term which remits to a Greek origin: thanatos meaning death and logy meaning study. This way the discussed subject is wide however it refers only to human death. The purpose of this work is through a literature review give an actualization dates to law professionals, as well as to those who work in peritial activity in Medicine and Dentistry, searching to approach questions referred to death diagnostic and its certification, its law aspects and details referred to peritial tests in Tanatology

Forensic tanatology: biological and legal aspects

ensic tanatology is a term which remits to a Greek origin: thanatos meaning death and logy meaning study. This way the discussed subject is wide however it refers only to human death. The purpose of this work is through a literature review give an actualization dates to law professionals, as well as to those who work in peritial activity in Medicine and Dentistry, searching to approach questions referred to death diagnostic and its certification, its law aspects and details referred to peritial tests in Tanatology

Validation of a physical anthropology methodology using mandibles for gender estimation in a Brazilian population

Validation studies of physical anthropology methods in the different population groups are extremely important, especially in cases in which the population variations may cause problems in the identification of a native individual by the application of norms developed for different communities. OBJECTIVE: This study aimed to estimate the gender of skeletons by application of the method of Oliveira, et al. (1995), previously used in a population sample from Northeast Brazil. MATERIAL AND METHODS: The accuracy of this method was assessed for a population from Southeast Brazil and validated by statistical tests. The method used two mandibular measurements, namely the bigonial distance and the mandibular ramus height. The sample was composed of 66 skulls and the method was applied by two examiners. The results were statistically analyzed by the paired t test, logistic discriminant analysis and logistic regression. RESULTS: The results demonstrated that the application of the method of Oliveira, et al. (1995) in this population achieved very different outcomes between genders, with 100% for females and only 11% for males, which may be explained by ethnic differences. However, statistical adjustment of measurement data for the population analyzed allowed accuracy of 76.47% for males and 78.13% for females, with the creation of a new discriminant formula. CONCLUSION: It was concluded that methods involving physical anthropology present high rate of accuracy for human identification, easy application, low cost and simplicity; however, the methodologies must be validated for the different populations due to differences in ethnic patterns, which are directly related to the phenotypic aspects. In this specific case, the method of Oliveira, et al. (1995) presented good accuracy and may be used for gender estimation in Brazil in two geographic regions, namely Northeast and Southeast; however, for other regions of the country (North, Central West and South), previous methodological adjustment is recommended as demonstrated in this study

Human identification analysis using PCR from the root portion of dental elements under different conditions of temperature and exposure time

Introduction and objective: The main exogenous factors limiting the retrieval of information from human remains are fire and accidents involving high temperatures. Teeth, due to their relatively high degree of chemical and physical resistance, offer the possibility for the recovery of genetic material, becoming important in forensic cases. With the aim to contribute to a standardization of the protocols employed in DNA extraction and analysis, it was evaluated the integrity of DNA recovered from dental roots submitted to high temperatures, simulating what happens to burnt people. Material and methods: Extractions of genomic DNA were made from the dental root after exposure to high temperatures (600ºC, 800ºC and 1000ºC), during 10, 30 and 60 minutes. Results and conclusion: After molecular analysis through PCR technique, it was verified that DNA amplification of the samples was not possible at any of the periods and temperatures analyzed