No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.

Forensic STR analysis is limited by the quality and quantity of DNA. Significant damage or alteration to the molecular structure of DNA by depurination, crosslinking, base modification, and strand breakage can impact typing success. Two methods that could potentially improve STR typing of challenged samples were explored: an in vitro DNA repair assay (PreCR™ Repair Mix) and whole genome amplification. Results with the repair assay showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally-damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR™ assay. The data suggest that the use of PreCR™ in casework should be considered with caution due to the assay’s varied results. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. Several modifications of the original DOP-PCR primer were evaluated. These modifications allowed for an overall more robust amplification of damaged DNA from both contemporary and historical skeletal remains compared with that obtained by standard DNA typing and a previously described DOP-PCR method. These new DOP-PCR primers show promise for WGA of degraded DNA.

Made available in DSpace on 2016-08-10T10:38:39Z (GMT). No. of bitstreams: 1 Ian Marques Candido.pdf: 1079357 bytes, checksum: dac0a34c413d6875a597e0f2ba2a41f6 (MD5) Previous issue date: 2013-02-08 / The identification of human remains in decomposition, charred, skeletal remains and mass disasters can be performed by Forensic Genetics and, in most cases, bones and teeth are the only viable source for DNA typing .Thus, considering the large number of bones used in human identification and the need for standardization of DNA extraction in this kind of sample, the aim of this study is to compare two techniques of DNA extraction, with the possibility of automation. The analysis was performed on twenty five human bones evaluating the quantity of the extracted genetic material, genetic profiles obtained for each sample and the time analysis by method used. With magnetic bead in platform automated, analysis time was 3 hours to process 12 samples, whereas by silica column four samples in 27 hours. Magnetic bead recovered a larger amount of DNA in 88% of samples. 68% of the samples magnetic particle had a high amplification partial (9/16 loci) and silica column only 36%. Therefore, the method used magnetic bead is suitable for automating the extraction processes. / A identificação humana de restos mortais em avançado estado de decomposição, carbonizados, desastres em massa e esqueletizados pode ser realizada pela Genética Forense e, na maioria das vezes, ossos e dentes são as únicas fontes de DNA viáveis para análise. Dessa maneira, considerando o grande número de ossos utilizados na identificação humana e a necessidade de padronização da técnica de extração de DNA nesse tipo de amostra, o objetivo do presente trabalho é comparar duas técnicas de extração de DNA, com possibilidade de automação. A análise foi realizada em vinte e cinco ossos humanos avaliando a quantidade do material genético extraído, os perfis genéticos obtidos em cada amostra e o tempo de análise gasto pela metodologia de extração. Com a metodologia de extração por partículas magnéticas utilizando plataforma automatizada, o tempo de análise foi de 3 horas para processar 12 amostras, enquanto que por coluna de sílica 4 amostras em 27 horas. Partícula magnética recuperou uma maior quantidade de DNA em 88% das amostras. 68% das amostras extraídas por partículas magnéticas tiveram uma amplificação parcial alta (9/16 loci) e por coluna de sílica apenas 36%. Por conseguinte, a metodologia de extração por partículas magnéticas é apropriada para a automatização dos processos de extração.

The ability of jurors and juries to comprehend and utilise scientific evidence in Australian criminal trials has been examined. From mock jury surveys relating to DNA profiling evidence, it was determined that most respondents were able to comprehend some basic and applied statistics, although their ability was in part related to their knowledge of English and their level of education. The point at which mock jurors were prepared to convict an accused solely on the basis of DNA profiling evidence was examined and found to be low compared with the strength of DNA profiling evidence commonly presented in Australian courts. Mock jurors also demonstrated the ability to process evidence that was presented in a Bayesian framework; commencing with prior odds, introducing new information and culminating in posterior odds. From a survey of Australian forensic scientists, including fraud investigators, it was found that most practitioners' concerns could be addressed by greater pre-trial consultation between experts and legal advocates. Improved knowledge within the legal profession concerning the jargon, principles, procedures, limitations and conclusions to be drawn from different scientific disciplines, prior to presenting this evidence in court, is recommended as the means by which complex evidence can be better adduced from expert witnesses and better presented to juries in criminal trials. Finally, from interviewing actual jurors in criminal trials in the Australian Capital Territory it was determined that where jurors' expectations of scientific evidence, particularly DNA profiling evidence, are not met, high levels of juror frustration and speculation may culminate in hung juries. The adversarial setting of criminal proceedings was also found to produce an environment in which jurors felt that information that would assist them in reaching a verdict was being deliberately withheld. The ability of the jury to ask questions and the allowed nature of those questions were also examined, with the resultant recommendation that juries be given more explicit information at the commencement of trials to inform them about their rights and obligations when asking questions.

[Truncated abstract] The collection of buccal cells is common practise in the epidemiological and forensic science. Unlike venipuncture collection of blood; it is a safer, non-invasive method for collection of biological material. The methods by which these cells are collected from the inner cheek of an individual and stored are the key elements in preserving DNA. Typically, forensic samples require long term storage. Samples are commonly collected on cotton swabs and stored moist at low to ultra-low temperatures (less than -20oC). Although this is the method of choice in most forensic facilities, there are drawbacks. The samples are inherently contaminated with microflora within the oral cavity and the moisture allows a plethora of microorganisms to grow. As the time frame that has elapsed from collection to storage increases, there is an exponential increase in bacterial cells. Storage of containers containing swabs coated with cells at temperatures below 20oC is also costly due to requirements for large freezers which are running and monitored over 24 hours. In the pass 10 to 15 years, researchers have focussed on alternative ways to store buccal cells. The FTA card system by Whatman is one such development. The FTA card is unique in that it provides a means for the collection of buccal cells for storage at room temperature. DNA profiling from samples stored in this way for 11 years has been successfully achieved. The filter paper matrix of the FTA card binds and subsequently lyses cells. ... (2) The second component of this thesis describes a study which subjected cells on buccal swabs to various conditions of increased temperature over periods of time to establish if DNA could be amplified. The aim was to mimic exposure to the vigours of field conditions, particularly in the extreme local environments that prevail in the United Arab Emirates. a. Initially, buccal cells stored at -20oC over 360 days were used to mimic standard archiving procedures. The cells were subsequently transferred to FTA cards, amplified and profiled by using ABI AmpFLSTR Identifiler PCR Amplification Kit (Applied Biosystems, Foster City, CA). Complete STR profiles were successfully recovered from the archived swabs. In most cases 100% of alleles were recovered, suggesting that it is feasible to transfer DNA from properly archived buccal swabs to FTA cards. b. The second phase involved the storage of fresh swabs that had been artificially aged by using incubation temperatures ranging from 40oC to 100oC. Partial profiles resulted from artificially aged samples, indicating that the prevailing conditions prior to low temperature storage of the swabs plays an important role in ensuring cellular integrity and thus, DNA quality. Results from this study suggest that it is possible for biological samples stored under correct conditions to be transferred from swabs to FTA card. In combination, the two chapters presented in this study show that it is feasible to transfer achieved forensic biology samples from swabs to the FTA card system. However, it is necessary to ensure that the samples are treated in the correct manner so as to minimise contamination from external sources and to maintain the correct environmental state to maintain intact cells and usable DNA.

DNA profiling has ignited public interest and consequently their expectations for the capabilities of forensic criminal and science investigations. The prospect of characterising the genetic makeup of individuals or trace samples from a wide variety of depositional and post-mortem circumstances raises the question of how reliable the methods are given the potential for prolonged exposure to variation in environmental factors, i.e. temperature, pH, UV irradiation and humidity, that are known to induce damage to DNA. Thus, it is crucial to verify the validity of the DNA profiling for characterising the genetic makeup of post-mortem tissues. This project aimed to assess the reliability of sequence and microsatellite based genotyping of tissues (muscle, hair and bone) sampled from carcasses over a two year post-mortem period. This assessment investigated the impact of environment induced DNA degradation in the local geographic region that is typical of the circumstances that confront forensic practitioners in southern Australia and to utilise rigorous controls by studying animals whose time of death and burial was known and for which we had pre-decay tissue samples available. A ‘body farm’ with 12 pig carcasses on the northern Adelaide plains, ~60km north of Adelaide, which has a typical southern Australian Mediterranean climate, i.e. cold wet winters and hot dry summers. Pigs (Sus scrofa) were used as an experimental analogue for human subjects because of the logistical and ethical reasons. The pig carcasses were allocated among three treatments: four were left on the surface, four were buried at 1m depth, and four were buried at 2 m depth. These ‘burial’ conditions mimic a range of conditions encountered typically in forensic and archaeological studies. Cortical bone samples were taken from each pig carcass at one week, one month, three months, six months, one year and two years post-mortem and muscle and hair over the same sampling period for as long as those tissue types were present. A set of PCR primers to amplify two (short and a long) fragments from the hypervariable part of the mitochondrial control region (HVRI) that is used in forensic and evolutionary studies of humans and many other mammal species were developed. Also a panel of four pig microsatellite loci with fluorescent labels to facilitate automated multiplex genotyping. These loci matched as closely as possible the core motifs and allele lengths typical of the commercially available microsatellite marker kits used in Australian forensic science labs so that our experiments were as good a model as possible of the human forensic DNA technology. In this study it was possible to retrieve samples from muscle tissue up to 90 days, hair up to one year and bone at two years post-mortem. The analyses showed that the long and short HVRI region PCR fragments were only amplifiable up to 30 days from muscle tissue and that these fragments were amplifiable up to one year from hair. In contrast, in cortical bone both PCR fragments were amplifiable up to two years. The long fragment disappeared in muscle tissue completely after 30 days and in hair after six months. However, the long fragment was present in cortical bone even at two years. Overall, there was a general trend of loss of concentration of both the long and short fragments over time. Comparisons of the HVRI nucleotide sequences among tissues sampled from individual animals showed substitution changes in muscles as early as 30 days (3 out of 6 individuals) and hair at six months (1 out of 6 individuals). In contrast, in cortical bone substitutions first appeared at 365 days (1 out of 6 individuals). The most common substitution observed in all tissues types was the C-T transition, with A-G transversions observed in two episodes and C-A transversion observed in one episode. Analyses of microsatellite genotypes in muscle tissues showed high allele peaks on chromatograms up to day seven samples. However, by three months PCR was not successful from muscle tissue. While, bone tissue had lower allele peak heights compared to the muscle tissues, alleles were detectable up to six months. Allele drop out occurred for one animal (at 2 meters) in muscle tissue at the dinucleotide locus and for another animal (kept on surface) also in muscle tissue at a tetranucleotide locus. Stuttering was observed for a single animal at dinucleotide locus in muscle tissue (buried sample 2 meter depth). No stuttering or allele drop outs were seen in the bone tissue. Overall the four loci completely disappeared after 30 days in muscle tissue and after 180 days in bone tissue. In summary, analyses showed that post-mortem DNA degradation was present in all the three tissue types (muscle, hair and bone). The types of damage identified were DNA fragmentation, nucleotide substitutions and DNA loss, which resulted in a diminished frequency of successful PCR for mitochondrial and nuclear markers over time and stuttering and allele drop out in microsatellite genotyping. In addition, two nucleotide substitutions were concentrated in ‘hotspots’ that correlate with sites of elevated mutation rate in vivo. Also the frequency of successful PCR of longer nuclear and mitochondrial PCR products declined markedly more quickly than for shorter products. These changes were first observed at much shorter post mortem intervals in muscle and much longer post mortem intervals in hair and bone tissue. When considering the carcass deposition treatments, tissues that were retrieved from buried carcases showed higher levels of DNA degradation compared to tissues retrieved from carcases left on the surface. Overall, muscle tissue is a good source for DNA analysis in immediate post mortem samples, whereas hair and bone tissue are good source for DNA analysis from older samples. When comparing the microsatellite genotyping and mtDNA analyses, mtDNA is a reliable source for DNA analysis from tissue recovered from bodies that had decayed for longer post-mortem durations such as months to years, whereas microsatellite genotyping gives reliable results for tissue from shorter post mortem intervals (hours to few days). Therefore it is recommended that when analysing mtDNA sequences, cloning and sequencing PCR products can help to identify the base pair substitutions especially for tissue retrieved from longer post mortem intervals. In addition, increasing the template DNA concentrations and "neutralising" co-extracted DNA inhibitors should be considered when dealing with tissue from longer post mortem intervals. Finally, the more stringent protocols used in ancient DNA studies should be considered when dealing with tissue with much longer post mortem intervals in forensic settings. / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

The mitochondrion is a tiny organelle that is the power supplier of the cell and vital to the functioning of the body organs. Additionally it contains a small circular genome of about 16 kb, present in many copies which makes the mitochondrial DNA more viable than nuclear DNA. Mitochondrial DNA is also maternally inherited and thus provides a direct link to maternal relatives. These two properties are of particular use for forensic samples, which only contain limited or degraded amounts of DNA, and for historical samples (ancient DNA). This thesis presents work on the mitochondrial DNA in the hypervariable regions (HV) I and II, in both contemporary and historical samples. Forensic genetics makes use of mitochondrial DNA analysis in court as circumstantial evidence, and population databases are used for the calculation of evidence value. Population samples (299) across Sweden have been analysed in order to enrich the EDNAP mtDNA database (EMPOP) (paper I). The application of mitochondrial DNA analysis allowed for analysis of historical skeletal remains: Copernicus, 1473-1543 (paper II), Karin Göring, 1888-1931 (paper III) and Medieval bones, 880-1000 AD, from a mass grave found in Sigtuna, Sweden (paper IV). The thesis also includes analyses of bones and teeth from the shipwrecked crew of the Vasa warship, 1628, samples from the Vasa museum, Stockholm, Sweden (paper V). Overall, the varying age of the samples and the different conservation environments (soil and water) accounted for variations in quality, but still allowed for successful DNA analysis.

A genética forense vem se desenvolvendo cada vez mais, com novas tecnologias e implementação de novos conjuntos de marcadores de DNA com maiores níveis de informatividade. Os marcadores genéticos são amplamente usados na identificação humana, pois permitem distinguir indivíduos com alta acurácia. Duas classes de marcadores muito utilizadas atualmente são os STRs (Short Tandem Repeats) e os SNPs (Single Nucleotide Polymorphisms). Os STRs são altamente informativos e, portanto, úteis para a prática forense. Kits mais novos como GlobalFiler (Thermo Fisher Scientific) e PowerPlex Fusion System (Promega) apresentam a análise de mais de 20 loci STRs de uma só vez. Já os SNPs, por possuírem sua informatividade mais reduzida (necessita de mais loci analisados), são menos utilizados, porém apresentam vantagem em amostras degradadas de DNA; assim, conjuntos de identificação como o 52-plex desenvolvido pelo consórcio SNPforID e o conjunto IISNPs, vêm sendo estudados em várias populações do mundo. Com o desenvolvimento de técnicas de sequenciamento de nova geração (NGS Next Generation Sequencing) para análise de DNA, a obtenção de perfis de DNA se tornou mais acurada. Algumas plataformas permitem gerar perfis de até 96 indivíduos simultaneamente. Este estudo tem por objetivo principal analisar 171 marcadores genéticos (Amelogenina, Y-INDEL, 30 STRSs e 139 SNPs) em 340 indivíduos miscigenados da região da cidade de Ribeirão Preto (SP) utilizando a plataforma de sequenciamento de nova geração MiSeq Personal Sequencer (Illumina Inc.), bem como calcular as frequências alélicas e genotípicas, verificar a aderência ao equilíbrio de HardyWeinberg e estimar parâmetros forenses para os diferentes conjuntos de marcadores. Análises de ancestralidade foram realizadas para os conjuntos de SNPs. Para o preparo das bibliotecas de amostras a serem sequenciadas, foi utilizado o kit HaloPlex (Agilent Technologies, Inc), onde foram incluídos os marcadores dos kits GlobalFiler e PowerPlex Fusion System, e os SNPs existentes no conjunto do consórcio SNPforID (52-plex) e IISNPs (92 SNPs). De todos os marcadores incluídos no ensaio, apenas um SNP (rs763869) presente no conjunto SNPforID não pôde ser analisado devido a questões técnicas. Dos 139 SNPs analisados apenas seis apresentaram desvios significativos em relação ao equilíbrio de Hardy-Weinberg,número este esperado devido ao acaso. Os conjuntos de SNPs apresentam elevada informatividade com Probabilidade de Match de 6,48 x 10-21 (52-plex) a 4,91 x 10-38 (IISNP), e Poder de Exclusão de 0,9997 (52-plex) e 0,99999997 (IISNP). De modo geral, as inferências de ancestralidade obtida utilizando estes conjuntos, indicaram elevada contribuição europeia (superior a 70%) e baixa contribuição ameríndia (inferior a 10%) na população, enquanto que as análises de mistura individual se mostraram consistentes, com a maioria dos indivíduos apresentando elevada ancestralidade europeia. Os resultados dos marcadores relativos ao sexo (Amelogenina, Y-INDEL e DYS391) foram consistentes com o sexo dos doadores das amostras. As frequências alélicas e parâmetros forenses foram calculados para os STRs, revelando uma alta informatividade. A Probabilidade de Match combinada e o Poder de Exclusão combinado foram de 1,19 x 10-36 e 0,999999999997 respectivamente. Dos 29 STRs autossômicos presentes, seis apresentaram desvios ao equilíbrio de Hardy-Weinberg, refletindo possíveis falhas no sequenciamento e genotipagem destes marcadores / The field of forensic genetics has developed increasingly with the implementation of new sets of DNA markers with higher levels of informativeness. The genetic markers are widely used in human identification as they allow distinguishing individuals with high accuracy. Two of the most commonly used markers are the Short Tandem Repeats (STRs) and the Single Nucleotide Polymorphisms (SNPs). Newer kits such as GlobalFiler (Thermo Fisher Scientific) and PowerPlex Fusion System (Promega) can analyze more than 20 STRs loci at once. When comparing with STRs, the SNPs are less informative and many more loci are needed to reach the same informativeness of STR kits. However, they are advantageous when using degraded DNA samples. The identification sets such as the 52-plex developed by the SNPforID Consortium and the IISNPs have been analyzed in many worldwide populations. With the development of next generation sequencing techniques (NGS Next Generation Sequencing), obtaining DNA profiles has become more accurate and some platforms allow generating profiles of up to 96 individuals simultaneously. The main goal of this study is to analyze 171 markers (Amelogenin, Y-INDEL, 30 STRs and 139 SNPs) in 340 admixed individuals from Ribeirão Preto, SP, using the NGS platform MiSeq Personal Sequencer (Illumina Inc.). This will allow the calculation of allele and genotype frequencies, the verification of adherence to Hardy-Weinbergs equilibrium and the estimation of forensic parameters for each set of marker. Ancestry analysis was performed for the sets of SNPs. The HaloPlex kit (Agilent Technologies, Inc) was used for library preparation including the STRs from the kits GlobalFiler and PowerPlex Fusion System and the SNPs from the SNPforID consortium (52-plex) and IISNPs (92 SNPs) identification sets. A single SNP (rs763869) from the SNPforID set was not analyzed due to technical issues. Only six of the 139 analyzed SNPs presented significant deviation from the Hardy-Weinberg equilibrium expectations, which is expected by chance alone. The SNPs sets exhibited high informativeness, with matchprobability ranging from 6.48 x 10-21 (52-plex) to 4.91 x 10-38 (IISNPs) and exclusion power of 0.9997 (52-plex) and 0.99999997 (IISNPs). In general, ancestry estimates obtained using these sets indicated a high European contribution (higher than 70%) and low Amerindian contribution (less than 10%) in the population sample, while the individual admixture analyses exhibited were highly consistent, with the majority of individuals presenting high European ancestry. The results of the sex markers (Amelogenin, Y-INDEL and DYS391) were in agreement with the reported sexes from sample donors. The allele frequencies and forensic parameters calculated for the STRs revealed high informativeness. The combined match probability and the combined exclusion power were 1.19 x 10-36 and 0.999999999997 respectively. Six of the 29 autosomal STRs presented significant deviations from the HardyWeinberg equilibrium expectations, reflecting possible failures in sequencing and genotyping of these markers