Caracteriza????o de sistemas multiplex de miniSTRs e SNPs para an??lise de DNA degradado na casu??stica forense

• Autor(a) principal: Michelin, Katia
• Data de Publicação: 2011
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UCB
• Texto Completo: https://bdtd.ucb.br:8443/jspui/handle/tede/2702
• topic: DNA degradado; Gen??tica forense; Forensic genetics; Degraded DNA; MiniSTRs; SNPs; CNPQ::CIENCIAS BIOLOGICAS

Forensic Genetics, whose foundations date back to the discovery and characterization of the first hypervariable loci in 1984, is today one of the main areas of Criminalistics. STRs are now the basis for genetic identification in forensics and the DNA databases and allow DNA evidence analysis with high sensitivity and relative resistance to degradation. However, in cases where the forensic trace is extensively degraded, in limiting amounts or in the presence of inhibitors, results may not be enough to secure identification. In order to overcome these limitations, recent research has focused the search for markers that make it possible to amplify fragments of short sizes. Among the markers that were most successful in this strategy are the STRs themselves, new loci with shoterr and redesigned primers, and single nucleotide polymorphisms, or SNPs. In this study, we selected ten SNPs, part of a set of 52 loci developed by an international consortium, and nine miniSTRs, part of a set of 26 non- CODIS loci, eight autossomal loci and one gender marker. The markers selected were optimized for use in multiplex and genotyped in two hundred Brazilian population samples. They were also tested on artificially degraded samples in different size ranges and against three of the main inhibitors observed in the forensic casework, and determination of sensitivity, using control DNA samples successively diluted. Alternative protocols were tested for amplification, using commercial reagents and different temperatures for thermocycling programs. The results with the SNPs were quite satisfactory when amplified with the Multiplex PCR kit (Qiagen??), achieving good performance with degraded samples and also regarding inhibition and sensitivity. Some difficulties were observed, however, for the reliable determination of the genotype with the detection method chosen, the single base extension. These difficulties may explain some observations in population studies, which revealed a locus with heterozygosity higher than expected for the Hardy-Weinberg Equilibrium (HWE) and another locus in linkage disequilibrium with three others. Regarding miniSTRs, all loci analyzed are in the HWE and show no significant linkage disequilibrium. Resistance to inhibition varied with the protocols used, and was considerably improved using the commercials GoldStar?? Buffer kit and Multiplex PCR (Qiagen??). The best sensitivity was observed with the 34 cycles "Low Copy Number" protocol. Resistance to degradation was satisfactory, especially when compared to commercial kits AMPFlSTR?? Identifiler?? and PowerPlex ??? 16 System, although variable in different samples undergoing the same process of degradation. The causes of this variation could not be explained in this study and need to be further investigated. The results suggest that the use of non-CODIS miniSTRs and SNPs proposed may result in further relevant information in cases where the DNA is extensively degraded, in low copy number or in analysis that require a complex family reconstructions, such as in mass disasters victims identification. Additional validations are, however, necessary.