Análise de polimorfismos do DNA mitocondrial em indivíduos residentes na grande São Paulo para aplicação na identificação humana
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2010-06-30
Autores
Paneto, Greiciane Gaburro [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A identificação humana por meio da análise de DNA utiliza o perfil genético de um indivíduo baseado na combinação de diversos marcadores que são herdados de seus progenitores. Estes marcadores são geralmente diferenças nas sequências de DNA nuclear entre os indivíduos (polimorfismos). Em alguns casos, entretanto, a análise do DNA nuclear não pode ser aplicada. Isso ocorre quando o DNA da amostra apresenta-se degradado ou em casos onde o material biológico não apresenta o DNA nuclear. Nestes casos, a análise do DNA mitocondrial (DNA mt) é o método de escolha. Entretanto, em um mesmo indivíduo podem existir populações de DNA mt diferentes, fenômeno denominado heteroplasmia. Este trabalho teve como objetivo o estudo de polimorfismos presentes no DNA mt de indivíduos residentes na Grande São Paulo para aplicação na Identificação Humana. Para isso, foi sequenciada toda a região hipervariável do DNA mt de 160 indivíduos. Além disso, o SNP 3010 foi analisado por discriminação alélica (PCR em tempo real) para estudo do aumento do poder discriminatório quando os indivíduos não puderam ser diferenciados pela análise da região hipervariável. Foi desenvolvido, também, uma reação de SNaPshot em multiplex contendo 42 SNPs que permitiram a classificação das amostras em haplogrupos do DNA mt. Por fim, foram analisadas 100 amostras de cabelo dos mesmos indivíduos para o estudo da frequência de heteroplasmia na região HV3 do DNA mt. De um total de 160 amostras, 144 haplótipos diferentes foram encontrados quando analisamos toda a região hipervariável do DNA mt; 131 haplótipos foram únicos. O SNP 3010 foi suficientemente discriminatório para conseguir distinguir indivíduos que apresentavam o mesmo haplótipo em dois dos treze casos que apresentavam mais de um indivíduo com o mesmo haplótipo
The human identification by DNA analysis uses the genetic profile of an individual based on the combination of diverse markers that are inherited of its ancestors. These markers are generally differences in sequences of nuclear DNA between individuals (polymorphisms). In some cases, however, the analysis of the nuclear DNA cannot be applied. It occurs when the DNA sample is degraded or in cases which the biological material does not content nuclear DNA. In these cases, the mitochondrial DNA (mtDNA) analysis is the choice method. However, inside one individual can exist different mtDNA populations, phenomenon called heteroplasmy. The aim of this work was to study mtDNA polymorphisms in individuals residents in São Paulo metropolitan area for application in Human Identification. For this, we have sequenced the entire hypervariable region of mtDNA for 160 individuals. Furthermore, the SNP 3010 was analyzed by allelic discrimination (Real-Time PCR) to study the increase of the discriminatory power when individuals could not be differentiated by analysis of the hypervariable region. It was developed also, a SNaPshot multiplex reaction containing 42 SNPs that allowed the classification of samples in mtDNA haplogroups. Finally, 100 hair samples, from the same individuals, were analyzed for the study of heteroplasmy frequency in the HV3 region of mtDNA. Among 160 samples, 144 different haplotypes were found when the entire hypervariable region of mtDNA was analyzed; 131 haplotypes were unique. The SNP 3010 SNP was able to distinguish individuals who had the same haplotype in two of thirteen cases with more than one individual with the same haplotype. Our population was classified in 46.6% of African, 27.3% of European, 25.5% of Native American and 0.6% Asian origin using the SNaPshot panel of 42 SNPs in multiplex
The human identification by DNA analysis uses the genetic profile of an individual based on the combination of diverse markers that are inherited of its ancestors. These markers are generally differences in sequences of nuclear DNA between individuals (polymorphisms). In some cases, however, the analysis of the nuclear DNA cannot be applied. It occurs when the DNA sample is degraded or in cases which the biological material does not content nuclear DNA. In these cases, the mitochondrial DNA (mtDNA) analysis is the choice method. However, inside one individual can exist different mtDNA populations, phenomenon called heteroplasmy. The aim of this work was to study mtDNA polymorphisms in individuals residents in São Paulo metropolitan area for application in Human Identification. For this, we have sequenced the entire hypervariable region of mtDNA for 160 individuals. Furthermore, the SNP 3010 was analyzed by allelic discrimination (Real-Time PCR) to study the increase of the discriminatory power when individuals could not be differentiated by analysis of the hypervariable region. It was developed also, a SNaPshot multiplex reaction containing 42 SNPs that allowed the classification of samples in mtDNA haplogroups. Finally, 100 hair samples, from the same individuals, were analyzed for the study of heteroplasmy frequency in the HV3 region of mtDNA. Among 160 samples, 144 different haplotypes were found when the entire hypervariable region of mtDNA was analyzed; 131 haplotypes were unique. The SNP 3010 SNP was able to distinguish individuals who had the same haplotype in two of thirteen cases with more than one individual with the same haplotype. Our population was classified in 46.6% of African, 27.3% of European, 25.5% of Native American and 0.6% Asian origin using the SNaPshot panel of 42 SNPs in multiplex
Descrição
Palavras-chave
Polimorfismo (Genetica), DNA, Heteroplasmia, Mitochondrial DNA, Polymorphisms, Heteroplasmy
Como citar
PANETO, Greiciane Gaburro. Análise de polimorfismos do DNA mitocondrial em indivíduos residentes na grande São Paulo para aplicação na identificação humana. 2010. 78 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas, 2010.