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In forensic casework it is often necessary to obtain genetic profiles from crime scene samples that contain increasingly smaller amounts of genetic material, called Low Template DNA (LTDNA). Two examples of LTDNA sources are touch DNA evidence and dust bunnies. Touch DNA refers to DNA that is left behind through casual contact of a donor with an object or another person. Touch DNA can be used to prove a suspect was present at a crime scene. Dust bunnies, or dust conglomerates, typically contain trapped shed skin cells of anyone in the vicinity along with fibers, dirt, hair, and other trace materials. Dust specimens are a potential source of forensic evidence that has been widely underutilized in the forensic community. This is unfortunate because a dust bunny could not only be used to associate a person or crime scene – through trace materials such as fibers – but also to positively identify – through a DNA profile. For example, if a dust specimen is found on a piece of evidence suspected of being moved from its original location, for instance as a body that is too heavy to carry and therefore collects dust while being dragged, then it could be used to link a suspect to a crime scene. Standard methods for obtaining and analyzing touch DNA have been established, but the techniques are not ideal. First, by nature, the 'blind-swabbing' technique, which involves cotton swabs or adhesive tape being applied to an area of interest, can artificially create mixtures of biological material that was originally spatially separated. Second, because the amount of DNA present is typically very low, standard analysis methods may not be sensitive enough to produce probative profiles. In the case of mixtures, the minor component's DNA may go undetected. Dust specimens contain degraded genetic material that has been accumulating for an unknown amount of time. Additionally, dust is usually a conglomeration of genetic material from multiple donors so a mixed profile, if any, is likely to be recovered if standard analysis methods are used. In order to overcome these obstacles presented by LTDNA, a micro-manipulation and combined cell lysis/direct PCR amplification technique has been developed that is sensitive enough to obtain full or probative STR profiles from single or clumped bio-particles collected from touch DNA and dust evidence. Sources of touch DNA evidence such as worn clothing items, touched objects, and skin/skin mixtures are easily sampled using an adhesive material on a microscope slide. Dust specimens can be dispersed onto an adhesive material as well. Targeted bio-particles are then "picked" with a water-soluble adhesive on a tungsten needle and deposited into a micro-volume STR amplification mix. Individual selection and analysis of isolated bio-particles reduces the chance of mixed profile recovery. To aid in the release of genetic material present in the bio-particles, a lysis mix containing a thermostable proteinase is then added to the sample. Samples are then analyzed using standard capillary electrophoresis (CE) methods. In addition to identifying the donor source of these LTDNA sources, it would be beneficial to a criminal investigation to identify the tissue source of the biological material as well. While it is widely speculated that the material originates from shed skin cells, there is little confirmatory evidence proving this assertion. Knowledge of the nature of the evidence could be vital to prevent its misinterpretation during the investigation and prosecution of a crime. Here tissue specific mRNA biomarkers have been evaluated for their use in tissue source determination using a highly sensitive High Resolution Melt (HRM) temperature assay that detects the selectively amplified targets based on their melt temperatures. Using the enhanced genetic analysis technique described above, DNA profile recovery has been markedly enhanced in sources of Touch DNA evidence and dust specimens compared to standard methods. Additionally, the molecular-based characterization method could potentially provide a better understanding of the meaningfulness of the recovered DNA profiles. This optimized strategy provides a method for recovering highly probative data from biological material in low template samples in an efficient and cost effect manner.

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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciência da Saúde, 2014. / Submitted by Ana Cristina Barbosa da Silva (annabds@hotmail.com) on 2015-01-22T16:24:20Z No. of bitstreams: 1 2014_ArthurEstivaletSvidzinski.pdf: 3625845 bytes, checksum: afc856ef59683659e917b21d2b7e4d7c (MD5) / Approved for entry into archive by Ruthléa Nascimento(ruthleanascimento@bce.unb.br) on 2015-02-11T18:36:28Z (GMT) No. of bitstreams: 1 2014_ArthurEstivaletSvidzinski.pdf: 3625845 bytes, checksum: afc856ef59683659e917b21d2b7e4d7c (MD5) / Made available in DSpace on 2015-02-11T18:36:28Z (GMT). No. of bitstreams: 1 2014_ArthurEstivaletSvidzinski.pdf: 3625845 bytes, checksum: afc856ef59683659e917b21d2b7e4d7c (MD5) / Genotipagem obtidas da analise da sequencia de DNA pelas mais diversas metodologias, os perfis de DNA, tem sido utilizadas frequentemente como ferramentas de investigação criminal e produção de provas periciais. Esses perfis tem sido cada vez mais solicitados as pericias, mesmo em situações onde a quantidade de DNA presentes nas amostras é muito pequena. Este é o caso de amostras produzidas por contato, o touch DNA, onde objetos manipulados ou que entraram em contato com a pele humana apresentam pequenas quantidades de células depositadas em sua superfície, que podem ser alvos de pesquisa de material genético. Neste tipo de amostra, uma grande dificuldade é identificar as melhores regiões para coleta, uma vez que o material depositado por contato não é visualmente destacado. A pesquisa de impressões digitais utiliza pós reveladores com a finalidade de revelar impressões digitais latentes, ou seja, possibilitam identificar as áreas de um objeto que entraram em contato com a pele humana. O objetivo deste trabalho foi avaliar a utilização de pós reveladores como ferramentas de triagem para coleta de material biológico deixado por contato com a finalidade de obtenção de perfis genéticos. Em primeiro lugar foi avaliada a quantidade de pó aderido a impressões deixadas por diferentes partes das mãos e a correlação entre a quantidade de pó aderido e a quantidade de DNA recuperado em uma amostra produzida por contato. A seguir foi avaliada a influência do pó revelador nos procedimentos laboratoriais envolvidos em uma análise de DNA. Para tanto, foi observado o efeito inibidor dos pós reveladores sobre a reação de PCR, a capacidade de diferentes métodos de extração em diminuir essa inibição e o resultado de um procedimento de purificação sobre o efeito inibitório. Finalmente, foi feita a pesquisa de DNA em cinco objetos relacionados a ocorrências criminais: arma de fogo, cartucho de munição, faca de cozinha, volante de veículo e alavanca de câmbio. Os resultados mostraram que a quantidade de pó aderido variou conforme a região da mão que produziu a impressão e que foi diretamente relacionada com a quantidade de DNA presente na amostra. O pó revelador apresentou um importante efeito inibidor sobre a reação de PCR. Os métodos de extração de DNA diminuem o efeito inibitório, porém este continua relevante. O procedimento de purificação não eliminou totalmente o efeito inibitório, porém o reduz a uma intensidade em que não comprometeu totalmente a obtenção de perfis genéticos. Nos cinco objetos testados foi possível coletar DNA em grande quantidade no volante de veículo e em quantidades razoáveis na arma de fogo, alavanca de câmbio e faca de cozinha. O cartucho de munição apresentou uma quantidade de DNA recuperada muito baixa, insuficiente para a produção de perfis genéticos de qualidade. Portanto, podemos concluir que pós reveladores podem ser utilizados como ferramentas para a eleição das regiões de coleta de material biológico produzido por contato. Mesmo considerando o seu efeito inibitório sobre a reação de PCR, amostras coletadas juntamente com pós reveladores são capazes de produzir perfis genéticos com qualidade suficiente para análise genética. __________________________________________________________________________________ ABSTRACT / DNA analysis has been used as a tool of criminal investigation and for producing forensic evidence. This kind of exam has been increasingly requested, even in situations where the amount of DNA present in the samples is very small. For example, samples of touch DNA, which consists of objects manipulated or touched by human skin that have small amounts of cells deposited on its surface, potential targets to DNA analysis. In this kind of sample, a great challenge is to identify the best locations for sample collection, since the biological material is not easy to identify. The fingerprint revelation technique uses fingerprint powders to reveal latent fingerprints allowing the identification surfaces touched by human skin. The aim of this study was to evaluate the possibility of using the fingerprint powders as screening tools for collecting biological material in touch DNA samples, obtaining good quality genetic profiles. The first step was to study the amount of powder adhered to the impressions produced by the different parts of the hands and the correlation between the amount of powder and the amount of DNA recovered. Next, the influence of fingerprint powder on laboratory procedures involved in DNA analysis was evaluated. The inhibitory effect of fingerprint powder in the PCR reaction was tested followed by the effect of different extraction methods on the inhibition. One DNA purification step was even included to improve the results. Finally, five different objects related to crimes: firearms, ammunition cartridge, kitchen knife, steering wheel and gear shift were proceeded to DNA analysis. The results showed that the amount of fingerprint powder recovered differed according the region of the hand which produced the fingerprint and the amount of adhered powder was directly related to the amount of DNA obtained. The fingerprint powder had a significant inhibitory effect on the PCR reaction. The methods of DNA extraction decreases the inhibitory effect, but it remains relevant. The purification procedure does not completely eliminate the inhibitory effect, but reduces an intensity that does not completely compromise the obtaining genetic profiles. In the five tested objects, it was possible to collect good amounts of DNA in the steering wheel, and in reasonable quantities in the firearms, gear shift and kitchen knife. The cartridge ammunition was the only object which was not possible to recover DNA in sufficient amount to produce quality genetic profiles. Therefore, we conclude that fingerprint powders can be used as tools for choosing the right regions to collect biological material in touch DNA samples. Even considering its inhibitory effect on the PCR reaction, samples collected along with fingerprint powders are able to produce genetic profiles with sufficient quality for genetic analysis.

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Locard’s exchange principle states that every contact leaves a trace. When someone touches a surface or object, they leave a touch deposit. Touch deposits are composed of epithelial cells and random debris from the toucher’s fingers that have been shed onto the contacted surface. This is useful when a fingerprint isn’t distinct enough to be collected; DNA could be recovered instead to create a genetic profile. This project focuses on discovering how cells in each touch deposit are suitable for genetic analysis using micromanipulation techniques and understanding patterns between type of touch and quality of genetic profile. The micromanipulation technique is done using a water-soluble adhesive and a tungsten needle to isolate and collect individual and grouped cells that might be useful for these types of samples. These tools under a microscope allow the skin cells to be separated from the larger sample and individually transferred into tubes for downstream biochemical reactions, including quantification and profiling. While micromanipulation is a technique that has been successful in methods involving epithelial cells, the validity of its use when applied to touch DNA cells, which are in less pristine condition than most samples due to the nature of touch deposits, is still being evaluated. The micromanipulation of grouped cells resulted in more probative genetic profiles and fewer drop-ins, despite inconclusive nucleation, in comparison to individually collected, nucleated cells.

A number of people have criticized the fact that courts often over-rely on DNA- evidence and that the shortcomings of DNA-analyses are rarely addressed. One flaw with DNA-analyses is that they cannot distinguish whether the presence of the DNA-trace has been caused by a so-called secondary transfer. Secondary transfer, the occurrence of which has been confirmed by several scientific stud- ies, means that DNA is transferred in two stages via contacts. A transfer of DNA can thus first occur from one person to another, for example by shaking hands, and then from the other person to a material, for example by grabbing a weapon. This poses a risk that DNA from an innocent person can be transferred through another person to a crime scene. A fundamental principle of criminal procedure is that the full burden of proof should be placed on the prosecution. This principle is derived from the presump- tion of innocence in Article 6.2 of the ECHR. As a consequence, the defendant has a right to remain silent throughout the judicial proceedings. Should the de- fendant instead be required to explain some of the circumstances of the case, a burden of proof would fall on the defendant, a so-called burden of explanation. Thus, a requirement that the defendant explain some of the circumstances of the case conflicts with the principle of the prosecutor's full burden of proof. Given the criticism that the courts rarely consider the shortcomings of DNA- evidence, there is a risk that the responsibility falls on the defendant to point out that a secondary transfer of DNA has occurred and to actively work towards convincing the court of his or her innocence. The purpose of the paper is thus to investigate the courts' consideration of these objections from the accused. The question is if a burden of explanation is placed on the accused and whether this is compatible with the presumption of innocence. To fulfill the purpose, an empirical study was conducted in the form of an analysis of 16 district court and appellate court decisions. The results showed that the defendant was imposed a burden of explanation in several cases. An exami- nation of the case law of the ECHR showed that there is, in exceptional cases, a possibility to impose an explanatory burden on the accused. However, the cases in the study were not covered by the exception. In conclusion, the practical ap- plication of the burden of proof is not compatible with the presumption of in- nocence. There is thus a need to educate judges on the complex subject of DNA to draw attention to the drawbacks of the evidence and to correct the false per- ception of DNA evidence as infallible. Furthermore, there is a need for a new precedent from the Supreme Court clarifying how the burden of explanation should be applied in accordance with the ECHR.