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To further facilitate mitochondrial DNA (mtDNA) sequence analysis for human identity testing, a better understanding of its mutation rate is needed. Prior to the middle 1990's the mutation rate applied to a forensic or evolutionary analysis was determined by phylogenetic means, This method involved calculating genetic distances as determined by amino acid or DNA sequence variability within or between species. The mutation rate as determined by this method ranged from 0.025-0.26 nucleotide substitutions/ site/ myr (million years). With the recent advent of mtDNA analysis as a tool in human identity testing an increased number of observations have recently come to light calling into question the mutation rate derived from the phylogenetic method. The mutation rate as observed from forensic analysis appears to be much higher than that calculated phylogenetically. This is an area that needs to be resolved in human identity testing. Mutations that occur within a maternal lineage can lead to a possible false exclusion of an individual as belonging to that lineage. A greater understanding of the actual rate of mutation within a given maternal lineage can assist in determining criteria for including or excluding individuals as belonging to that lineage. The method used to assess the mutation rate in this study was to compare mtDNA sequences derived from the HVI and HVII regions of the D-loop from several different maternal lineages. The sequence information was derived from five unrelated families consisting of thirty-five individuals. One intergenerational mutational event was found. This derives to approximately 1.9 nucleotide substitutions/ site/ myr. This mutation rate was very consistent with several other similar studies. This increased mutation rate needs to be considered by forensic testing laboratories performing mtDNA sequence analysis prior to formulating any conclusive results.

This master thesis deals with forensic genetics. Primarily is forensic genetics put into the historical context. Then, there is described a biological material and the possibility of detecting biological traces. Further work is focused on the analysis of DNA, while there are elaborated in detail the techniques used as nucleic acid isolation, quantification of DNA, as well as types of polymerase chain reaction (PCR). Also the thesis deals with methods of obtaining DNA profiles - electrophoresis and sequencing and their variants. Finally, the thesis concern the issues of interpretation of forensic genetic data. Forensic biological exercises for high school students are processed in the didactic part. It has been tested in practice in biological seminars at Jiráskovo gymnázium in Náchod. Key words Forensic genetics, biological traces, didactics of biology

This master thesis deals with forensic genetics. Primarily is forensic genetics put into the historical context. Then, there is described a biological material and the possibility of detecting biological traces. Further work is focused on the analysis of DNA, while there are elaborated in detail the techniques used as nucleic acid isolation, quantification of DNA, as well as types of polymerase chain reaction (PCR). Also the thesis deals with methods of obtaining DNA profiles - electrophoresis and sequencing and their variants. Finally, the thesis concern the issues of interpretation of forensic genetic data. Forensic biological exercises for high school students are processed in the didactic part. It has been tested in practice in biological seminars at Jiráskovo gymnázium in Náchod. Key words Forensic genetics, biological traces, didactics of biology

Philosophiae Doctor - PhD / DNA profiling the male specific region on the Y-chromosome is fundamental to forensic practise. Its recognised as a powerful analytical tool for investigation of sexual assault when the DNA evidence is highly admixed. Standard practises for processing sexual assault evidence include physically separate the sperm cell from the female fraction using differential extraction followed by autosomal DNA profiling. However, under specific scenarios of assault physical separation may not be possible due to the nature of the evidence. The research presented in this thesis was focused on the development and validation of the UniQTyper™ Y-10 prototype for male specific DNA profiling. The prototype which contains 10 Y-STR markers was developed and validated to deliver a rapid and cost-effective system while maintaining a forensic applicable level of performance. An allelic ladder is produced with an allele cloning approach for which an overview of the workflow and technicalities presented herein is aimed to assists an efficient bulk production process. In a second component novel sequence variation was reported across 153 sequenced alleles and submitted to Genbank. In this output the Y-STR panel was perused beyond the scope of length polymorphisms. In a proof of concept, its potential to discriminate between shared allele sizes by characterizing sequence structure variations is discussed. In a final component we generate the largest Y-STR survey across South Africa to establish reference data and to comprehensively assess the forensic genetics parameters for the UniQTyper™ Y-10.

Orientador: Regina Maria Barretto Cicarelli / Banca: Joyce Aparecida Martins Lopes Ferraz / Banca: Rogério Nogueira de Oliveira / Resumo: A identificação humana por meio do DNA constitui um dos produtos mais revolucionários da Genética Moderna, tornando-se uma ferramenta indispensável na investigação criminal. Essa identificação é baseada no perfil genético do indivíduo, pela combinação de diversos marcadores herdados de seus progenitores. Os marcadores são, geralmente,e diferenças nas sequências de DNA nuclear entre os indivíduos (polimorfismos). Em alguns casos, em que a análise do DNA nuclear não puder ser aplicada, a alternativa de maior sucesso é a análise do DNA mitocondrial (DNAmt). As mitocôndrias são organelas intracelulares de dupla membrana presentes em todas as células nucleadas de mamíferos, com genoma extracromossômico separado e distinto do genoma nuclear, o DNA mt. A maioria dos laboratórios que utilizam tipagem do DNAmt baseiam-se nos polimorfismos presentes na sequência de nucleotídeos na região não codificadora (também conhecida como região controle, hipervariável, ou D-loop) do DNAmt. No entanto, a classificação em alguns haplogrupos pode não ser possível com base em dados apenas da região controle. Assim, estudos sugerem a necessidade de tipagem adicional de polimorfismos de nucleotídeos únicos (SNPs) em outras regiões do DNAmt, em especial, nos casos onde não é possível diferenciar os indivíduos apenas pela análise da região hipervariável. Este trabalho teve como objetivo analisar, analisar 30 (trinta) SNPs do DNAmt em amostras de indivíduos não relacionados, nascidos e residentes no Estado do Espírito Santo, permitindo a classificação das mesmas em haplogrupos e complementando os dados de SNPs da região controle do DNAmt obtidos em trabalho anterior no laboratório, para posterior utilização em casos forense. De um total de 100 amostras, foram encontrados 19 haplogrupos e a população estudada foi classificada conforme sua origem em: 43% africana, 30% europeia... / Abstract: Human identification through DNA is one of the most revolutionary products of Modern Genetics, making it an indispensable tool in criminal investigation. This identification is based on the genetic profile of the individual, the combination of several markers inherited from their parents. The markers are generally differences in nuclear and DNA sequences between individuals (polymorphisms). In some cases, the analysis of nuclear DNA cannot be applied, the most successful alternative is the analysis of mitochondrial DNA (mtDNA). Mitochondria are intracellular organelles with a double membrane present on all nucleated mammalian cells with separate and distinct extrachromosomal genome nuclear genome, the mt DNA. Most laboratories use typing based mtDNA polymorphisms in the nucleotide sequence in the noncoding region (also known as the control region, the hypervariable or D-loop) of mtDNA. However, in some haplogrupos classification may not be possible based on only control data region. Thus, studies suggest the need for additional typing single nucleotide polymorphisms (SNPs) in other regions of mtDNA, especially in cases where it is not possible to differentiate individuals only by the analysis of hypervariable region. This study aimed to analyze, analyze thirty (30) SNPs of mtDNA in samples of unrelated individuals born and living in the State of Espírito Santo, allowing their classification in haplogroups and complementing the SNPs data of mtDNA control region achieved in previous work in the laboratory, for later use in forensic cases. A total of 100 samples, 19 were found haplogroups and the sample were classified according to its origin: 43% African, 30% European, 26% Native American, and 1% Asian. The haplogroup was found more L3 having African origin. Some samples of previous work could not be correctly classified only with the sequencing of the control region of mtDNA, after analysis of 30... / Mestre

Abstract not available

DNA as forensic evidence is valuable in criminal investigations. Implementation of new, sensitive and fast technologies is an important part of forensic genetic research. This thesis aims to evaluate new sensitive methods to apply in forensic DNA analysis including analysis of old skeletal remains. In Paper I and II, two novel systems for analysis of STRs, based on the Pyrosequencing technology, are presented. In Paper I, Y chromosomal STRs are analysed. Markers on the male specific Y chromosome are especially useful in analysis of DNA mixtures. In Paper II, ten autosomal STRs are genotyped. The systems are based on sequencing of STR loci instead of size determination of STR fragments as in routine analysis. This provides a higher resolution since sequence variants within the repeats can be detected. Determination of alleles is based on a termination recognition base. This is the base in the template strand that is excluded from the dispensation order in the sequencing of the complementary strand and therefore terminates the reaction. Furthermore, skeletal remains are often difficult to analyse, due to damaging effects from the surrounding environment on the DNA and the high risk of exogenous contamination. Analysis of mitochondrial DNA is useful on degraded samples and in Paper III, mtDNA analysis of 700 years old skeletal remains is performed to investigate a maternal relationship. The quantity and quality of DNA are essential in forensic genetics. In Paper IV the efficiency of DNA isolation is investigated. Soaking skeletal remains in bleach is efficient for decontamination but result in a lower DNA yield, especially on pulverised skull samples. In conclusion, this thesis presents novel sequencing systems for accurate and fast analysis of STR loci that can be useful in evaluation of new loci and database assembly as well as the utility of mtDNA in forensic genetics.

The pathetic cry 'Who is my father?' has been asked time and again the world over. Discovery of paternity, linked as it is with the processes - legal and scientific - of establishing the alleged father's relationship on a balance of probabilities is a very real problem in the field of family law in South Africa. Blood tests have proved to be one aid in its solution. However, the application of such tests carry with them their own specific difficulties, most notable from the point of view of the lawyer is the extent of the authority of the court to order such tests, the interpretation of the test results and the role and emphasis that should be given to the results of the blood tests in the final determination of each case. Lawyers have to be wary and avoid falling into the trap of the layman who has the distinct tendency to accept unquestionably anything backed by scientific authority. / Thesis (LL.M.)-University of Durban-Westville, Durban, 1994.