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Abstract(s)
O perfil genético de cada indivíduo pode ser obtido através do estudo de vários marcadores polimórficos presentes no DNA nuclear, nomeadamente os STRs. Em Genética Forense, a quantidade de DNA pode variar consideravelmente entre diferentes amostras, factor que poderá comprometer a obtenção dos respetivos perfis genéticos.
Deste modo, torna-se indispensável determinar a quantidade de DNA presente nas amostras forenses através de uma metodologia de quantificação, recorrendo a alguns kits comerciais. Esta etapa permite avaliar se uma amostra pode ou não seguir para amplificação por PCR e, em caso afirmativo, qual o kit de amplificação e volume de DNA que devem ser utilizados, garantindo, assim, o sucesso dos resultados.
De todas as amostras analisadas no contexto forense, as degradadas constituem o maior desafio, devido à presença de DNA em quantidade e qualidade muitas vezes limitante. Para ultrapassar esse obstáculo, surgiu um novo kit de quantificação denominado Quantifiler® Trio DNA Quantification. Este kit apresenta características melhoradas comparativamente aos kits anteriores, possibilitando detetar quantidades de DNA mais reduzidas, bem como proporções de DNA masculino versus DNA feminino mais díspares em amostras de mistura. Como mais-valia, o kit permite avaliar o nível de degradação das amostras. Para esse efeito, deteta dois alvos localizados no DNA autossómico, um alvo de 80 pares de bases e um alvo de 214 pares de bases, sendo que a razão entre as concentrações destes dois alvos constitui o índice de degradação de cada amostra.
No Serviço de Genética e Biologia Forenses - Delegação do Centro do Instituto Nacional de Medicina Legal e Ciências Forenses, I.P., procedeu-se à validação interna do kit Quantifiler® Trio, com base no Procedimento Geral de Validação de Ensaios, documento elaborado pelo serviço que define todos os passos e estudos a realizar durante este processo. Para testar o desempenho do kit foram estudados os parâmetros de precisão, limiares analíticos, especificidade e contaminação. A par disso, foi igualmente avaliada a influência dos resultados de quantificação obtidos com o kit na determinação dos perfis genéticos de amostras de mistura com diferentes proporções, assim como de amostras com diferentes índices de degradação, recorrendo ao kit de amplificação GlobalFiler®.
Este estudo demonstrou uma grande precisão e sensibilidade do kit Quantifiler® Trio, associado à especificidade deste para o DNA humano. Por fim, a informação dada pelo índice de degradação revelou ser fundamental para prever o impacto que diferentes níveis de degradação poderão ter na obtenção de um perfil genético.
The genetic profile of each individual can be obtained through the study of several polymorphic markers in the nuclear DNA, mainly the STRs. In Forensic Genetics, the amount of DNA can vary considerably among different samples, a factor which may interfere with the achievement of the respective genetic profiles. Thus, it is essential to determine the amount of DNA present in forensic samples by a quantification methodology, using commercial kits. This step provides guidelines on whether or not a sample is suitable for PCR amplification and, if possible, which amplification kit and DNA volume should be used, therefore ensuring the success of the results. In all samples analyzed in forensic context, degraded samples represent a major challenge, due to the presence of DNA in often compromised quantity and quality. To overcome this obstacle, a new quantification kit called Quantifiler® Trio DNA Quantification was developed. This kit has improved features when compared to previous kits used, making it possible to detect smaller amounts of DNA and proportions of male DNA versus female DNA more discrepants in mixture samples. In addition, the kit enables to evaluate the level of degradation of samples. For this purpose, it detects two targets located in the autosomal DNA, a target of 80 base pairs and another of 214 base pairs. The ratio between the concentrations of these two targets is referred to as the degradation index of each sample. In the Forensics Genetic and Biology Service - Central Delegation of the National Institute of Legal Medicine and Forensic Sciences, I.P., the internal validation of Quantifiler® Trio kit was carried out, based on the General Procedure Testing Validation, a document developed in the service which defines all the steps and studies to be conducted during this process. The kit performance was tested based on the study of precision parameters, analytical thresholds, specificity and contamination. Furthermore, it was also evaluated the influence of the quantification results obtained with the Quantifiler® Trio kit in determining genetic profiles of mixture samples with different ratios, as well as samples with different levels of degradation, using the GlobalFiler® PCR Amplification kit. This study demonstrated a high accuracy and sensitivity of the Quantifiler® Trio kit, along with its specificity for human DNA. Finally, the information provided by the degradation index was found to be essential to predict the impact that different levels of degradation may have on obtaining a genetic profile.
The genetic profile of each individual can be obtained through the study of several polymorphic markers in the nuclear DNA, mainly the STRs. In Forensic Genetics, the amount of DNA can vary considerably among different samples, a factor which may interfere with the achievement of the respective genetic profiles. Thus, it is essential to determine the amount of DNA present in forensic samples by a quantification methodology, using commercial kits. This step provides guidelines on whether or not a sample is suitable for PCR amplification and, if possible, which amplification kit and DNA volume should be used, therefore ensuring the success of the results. In all samples analyzed in forensic context, degraded samples represent a major challenge, due to the presence of DNA in often compromised quantity and quality. To overcome this obstacle, a new quantification kit called Quantifiler® Trio DNA Quantification was developed. This kit has improved features when compared to previous kits used, making it possible to detect smaller amounts of DNA and proportions of male DNA versus female DNA more discrepants in mixture samples. In addition, the kit enables to evaluate the level of degradation of samples. For this purpose, it detects two targets located in the autosomal DNA, a target of 80 base pairs and another of 214 base pairs. The ratio between the concentrations of these two targets is referred to as the degradation index of each sample. In the Forensics Genetic and Biology Service - Central Delegation of the National Institute of Legal Medicine and Forensic Sciences, I.P., the internal validation of Quantifiler® Trio kit was carried out, based on the General Procedure Testing Validation, a document developed in the service which defines all the steps and studies to be conducted during this process. The kit performance was tested based on the study of precision parameters, analytical thresholds, specificity and contamination. Furthermore, it was also evaluated the influence of the quantification results obtained with the Quantifiler® Trio kit in determining genetic profiles of mixture samples with different ratios, as well as samples with different levels of degradation, using the GlobalFiler® PCR Amplification kit. This study demonstrated a high accuracy and sensitivity of the Quantifiler® Trio kit, along with its specificity for human DNA. Finally, the information provided by the degradation index was found to be essential to predict the impact that different levels of degradation may have on obtaining a genetic profile.
Description
Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
Keywords
DNA nuclear Quantificação Kit Quantifiler® Trio Kit GlobalFiler® Teses de mestrado - 2015