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In forensic genetics, the analysis of postmortem bones is one of the most challenging due to the low quantity of degraded endogenous DNA. The most widely used approach for sample preparation, in those cases, is pulverizing the bone. However, processing pulverized bone is extremely delicate, requiring strict laboratory conditions and operating procedures. In fact, several recent publications have focused on non-powder approaches. The objectives of this study were, thus, to validate a non-powder protocol for DNA extraction from forensic bones and an alternative pretreatment, large fragment demineralization (LFD). Thirty human femurs and tibiae received by the Legal Medicine Institute of Brescia, Italy, were included in the study. Bone powder and one transversal section of the diaphysis were sampled from each bone. DNA extraction from the powder was carried out using PrepFiler BTA (BTA), while the transversal section was submitted to the alternative demineralizing pretreatment (LFD) followed by DNA extraction using the QIAamp DNA Investigator. DNA extracts were assessed for human DNA quantity and degradation by means of a validated in-house qPCR assay and amplified with commercial kits. Inhibition assessment was carried out through Quality Sensor analysis using 24plex QS Kit. The differences in quantity, quality of human DNA, and number of alleles detected between both methods were comparable and not statistically significant. We propose the use of the LFD protocol as a complementary approach capable of confirming the genotypes or detect alleles not observed using BTA, without the need for pulverization.

The identification of human remains using DNA analysis can be extremely challenging and its success is certainly influenced by the time elapsed since death. In that context, intact teeth have been shown to be highly successful in DNA analysis. However, restored teeth are usually available and, surprisingly, these specimens have been poorly studied. In fact, there are no reports regarding forensic DNA analysis of those types of samples in real cases. Therefore, the aim of this study was to perform DNA typing on healthy and restored teeth from exhumed human remains, which had been buried for 46 years. A powder-free DNA extraction protocol specifically designed for teeth was followed and human DNA quantitation and degradation assessment was performed using an in-house qPCR assay. Samples were amplified with commercial human identification kits for autosomal and Y chromosome markers. The obtained DNA profiles were compared to those of a previously processed femur sample as well as a buccal swab from a putative son. One healthy and one restored tooth yielded complete, concordant and compatible DNA profiles with previously typed samples from the femur and the putative son. Biostatistical calculations supported the paternity relationship with a likelihood ratio greater than 11 million. The present study highlights the use of restored teeth in a real exhumation case and the powder-free approach specifically designed for the extraction of DNA from teeth is discussed.

The typing of short tandem repeats (STRs) is a common method used in forensic laboratories based on its good performance. However, because of the relatively large size of amplicons (150–500 bp), most of them are unsuitable for degraded DNA and low copy number samples. So, there is a need to find new genetic makers with smaller amplicon size. Recently, some forensic researchers focused their attention to alternative and supplementary genetic markers in the human genome: insertion-deletion polymorphisms (InDels). They are diallelic, of smaller size, widely distributed throughout the genome and with a lower mutation rate compared to STRs. Due to their characteristics, InDels can aid forensic analysis even when a very small amount of DNA is available in a sample. In this study we tested the ability of a set of 38 InDel markers on genotyping different forensic samples containing low DNA concentration, already typed using the Identifiler kit but obtaining poor results. Statistical analysis was performed using the STRs results with appropriated softwares. After, InDel typing results were added to the STRs statistic calculations. The purpose of this work was to verify the actual usefulness of InDels analysis in order to improve the weight of the statistical analysis of challenging samples.

Teeth are widely used samples in forensic human genetic identification due to their persistence and practical sampling and processing. Their processing, however, has changed very little in the last 20 years, usually including powdering or pulverization of the tooth. The objective of this study was to present demineralized root tips as DNA sources while, at the same time, not involving powdering the samples or expensive equipment for teeth processing. One to five teeth from each of 20 unidentified human bodies recovered from midwest Brazil were analyzed. Whole teeth were demineralized in EDTA solution with daily solution change. After a maximum of approximately seven days, the final millimeters of the root tip was excised. This portion of the sample was used for DNA extraction through a conventional organic protocol. DNA quantification and STR amplification were performed using commercial kits followed by capillary electrophoresis on 3130 or 3500 genetic analyzers. For 60% of the unidentified bodies (12 of 20), a full genetic profile was obtained from the extraction of the first root tip. By the end of the analyses, full genetic profiles were obtained for 85% of the individuals studied, of which 80% were positively identified. This alternative low-tech approach for postmortem teeth processing is capable of extracting DNA in sufficient quantity and quality for forensic casework, showing that root tips are viable nuclear DNA sources even after demineralization.

For over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

Made available in DSpace on 2020-12-12T00:58:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Anaesthetics Research Society The use of Y-chromosomal genetic markers in forensic investigations demands the establishment of reliable and representative DNA databases of different reference populations. The genetic characterization of the Y chromosome variation in human populations requires the analyses of haplotype frequencies allied to haplogroup determination. The present study aimed to contribute to the Brazilian database by providing 1,382 Yfiler Plus individual profiles, from 11 Brazilian states. The Yfiler Plus markers showed high haplotype diversities in all Brazilian populations (>0.9970), allowing high intra-population discrimination in forensic investigations. Pairwise genetic distances showed a homogeneity between Brazilian populations (FST ≤ 0.0043; non-differentiation p-values ≥ 0.0212), indicating that admixed populations from Brazil can be represented in a single Yfiler Plus haplotype database, for forensic purposes. The performance of Haplogroup Predictor and NevGen software in haplogroup prediction based on Yfiler Plus and Yfiler haplotypes was evaluated in a subset of 416 Brazilian samples that were also genotyped for 51 Y-SNPs. In 25% of the samples, no classification or errors were found for at least one of the prediction tools or marker sets. NevGen presented lower error rates (5.52% and 8.65% with Yfiler Plus and Yfiler, respectively) than Haplogroup Predictor (16.11% with Yfiler Plus and 13.70% with Yfiler). In conclusion, both haplogroup prediction tools can be useful to direct the SNP typing, but present large error rates to be used in forensic analysis, especially in predicting African haplogroups in admixed South American populations. DNA Diagnostic Laboratory (LDD) State University of Rio de Janeiro (UERJ) Laboratory of Human and Molecular Genetics Pontifícia Universidade Católica do Rio Grande do Sul Laboratório de Biologia e DNA Forense Superintendência de Polícia Técnico-Científica do Estado de Goiás UNESP-Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas Laboratório de Investigação de Paternidade-NAC Coordenadoria de Perícias em Biologia Molecular POLITEC – Perícia Oficial e Identificação Técnica Faculdade Pitágoras Departamento de Medicina Legal Ética Médica e Medicina Social e do Trabalho da Faculdade de Medicina da USP Universidade de São Paulo (USP) IPATIMUP-Institute of Pathology and Molecular Immunology from the University of Porto I3S-Instituto de Investigação e Inovação em Saúde Universidade do Porto Postgraduate Program in Genetics and Molecular Biology Laboratory of Human and Medical Genetics Federal University of Pará Secretaria de Defesa Social Pernambuco Serviço de Endocrinologia e Metabologia do Hospital Universitário da Universidade Federal do Maranhão UNESP-Universidade Estadual Paulista Faculdade de Ciências Farmacêuticas Laboratório de Investigação de Paternidade-NAC Anaesthetics Research Society: 304413/2015-1

Blood Alcohol Concentration (BAC) refers to the amount of ethanol present in the blood of an individual. Ethanol is among the most common substances encountered in forensic toxicology because ethanol abuse is related to fatal traffic accidents and general anti-social behavior. The objective was to describe the profile of victims of fatal traffic accidents in the state of Mato Grosso between 2013 and 2015. We analyzed 3,045 case records from Mato Grosso state Official Public Forensic Institution, Politec. Positive results for the presence of ethanol in the blood of victims of fatal traffic accidents was higher for males 1,162 (83.1%), from 21 to 30 years of age, and occurred predominantly during the weekends. Traffic accidents in Cuiabá County had the highest mortality coefficient per 100 thousand inhabitants (28.9%) than the rest of the state. Regarding the months of the year, there was a homogenous distribution with a discrete predominance of cases in October, November and December. The incidents were more frequent between 22h, 8h and 11h/12h am. This study contributes not only for the identification of the individuals most likely to be involved in traffic accidents where ethanol was consumed, but also to subsidize public policies aimed towards intervening and raising awareness of damages due to association among driving and ethanol use or abuse.

The Applied Biosystems™ NGM Detect™ PCR Amplification Kit is a new Human Identification solution with increased sensitivity in comparison to NGM SElect Kit. The purpose of this study is to evaluate the performance of the NGM Detect™ kit compared to AmpFlSTR® NGM Select™ and AmpFlSTR® Identifiler® Plus kits, using a variety of casework samples. The study is being carried out on samples obtained from forensic cases such as sexual violence, exhumations, paternity, homicides and suspicious deaths. The sample typologies refer to: buccal swabs, nail swabs, swabs deriving from sexual violence, human biological tissues and liquids, clothes and objects of common use. The preliminary results show that the combined use of NGM Detect and NGM Select kits provide maximum information in a dual amplification strategy, as the size of several markers are complementary. Furthermore, the NGM Detect and NGM Select kits appear more sensitive than the Identifiler Plus kit. In conclusion, together with the NGM Select™ kit, the NGM Detect™ kit offers the possibility of recovering a lot of genetic information from degraded samples, delivering the best likelihood ratio value of identity compared to other ESS-focused solutions in the market today.

The discovery of human corpses in urban domestic settings does not constitute an unusual case in criminal casework. These scenarios can be very challenging to investigate since the uninformative evidences encountered also demand a multidisciplinary effort among several specialties in the forensic sciences field. The occurrence of this incident is usually accompanied by social isolation, which is an emblematic aspect of urban modern society. The elderly population is especially susceptible to being socially isolated, which is associated with higher mortality. We present a case report of an elderly woman who had been living with her husband's dead body, contributing to the scarce literature on the "Living with the Dead" phenomenon. The use of a multidisciplinary approach and the challenges that social isolation presents to forensic sciences and the contemporary society are discussed.