The purpose of this experiment was to determine if nucleic acid sequence based amplification (NASBA) is a suitable application for the differentiation of body fluids that might comprise a forensic evidence sample. NASBA is a sensitive RNA transcription based amplification system. NASBA could theorhetically be used for bodily fluid identification based upon amplification of tissue-specific mRNA transcripts present in a given forensic sample.Amplification of both Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Matrix Metalloproteinase 1 1 (MMPmRNA transcripts were used to determine that NASBA could amplify body fluid transcripts and whether it could distinguish between menstrual and non-menstrual blood, respectively. GAPDH is a housekeeping gene that is constituently expressed and its mRNA transcripts could therefore be used to determine whether non-menstrual blood could be amplified using the NASBA procedure. MMP 11 is a menstrual cycle-specific gene associated with endometrial breakdown. Using the mRNA transcripts from MMP 11, NASBA could be utilized for menstrual blood identification. In this study, non-menstrual and menstrual blood samples were analyzed with NASBA both in the presence and absence of chemical contamination. Contaminants utilized ranged from commercial automotive wax, transmission fluid, brake fluid, artificial tears, hand soap, 10% bleach, and the luminol blood detecting reagent. Non-menstrual blood was aliquoted onto a 1 cm x 1 cm cotton cloth for contamination, while menstrual blood was provided on a 1 cm x 1 cm area of sterile menstrual pad. All samples underwent Tri reagent extraction to obtain RNA samples for NASBA amplification.With respect to NASBA amplification data, non-menstrual blood data (from extracted RNA and unextracted blood samples) revealed the highest levels of amplification as shown in relative fluorescence units (RFU). Uncontaminated menstrual blood revealed the second highest amplification data. In the presence of chemical contamination, high levels of amplification were observed when samples were contaminated with brake fluid and commercial hand soap. Moderately low amplification was observed with samples contaminated with transmission fluid, 10% bleach, and artificial tears. NASBA amplification was completely inhibited in the presence of automotive wax and luminol. Cycle threshold (CO values for each amplification result were also obtained from each reaction. Smaller Ct values correspond to a higher NASBAreaction efficiency and therefore larger amplification values. The Ct values obtained for each amplified sample correlate strongly with the amount of amplification observed from reaction. Based upon the results of this experiment, NASBA should be considered as a novel tool for forensic evidence analysis. / Department of Biology

Nucleic Acid Sequence Based Amplification (NASBA) is a powerful in vitro technique for amplification. NASBA is routinely used in many fields of microbiology, including food microbiology, and most recently in the identification of forensic body secretions (saliva, tears, sweat, semen, vaginal secretions). NASBA has many advantages over the traditional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) including speed, high sample throughput and increased sensitivity. Proper selection of the sequence of importance and the designe of NASBA primers precisely for that sequence are the two most critical steps for any NASBA assay. Proper designe of NASBA primers includes important considerations such as product (amplicon) length, the addition of a T7 RNA polymerase promoter sequence at the 5’end of one primer, and a 3’AT rich region. Primer designing is, therefore, laborious and error-prone. Currently, no such software is available that facilitates primer designing for NASBA. In this study Java-based software for designing NASBA primers was developed which will enable rapid and specific NASBA primer designing for gene expression studies. The designed program focused on scripting minimum Java coding lines to reduce the time and storage space. Two codes were scripted for this software, a pseudo-code (for Java Program Developers) and Byte-code (for Windows operating system). Our results showed that Java is an efficient tool in searching sequences of interest within a gene (or mRNA), allowing for NASBA primers to be designed more quickly and effortlessly. The program has maximum memory storage capacity and allows the users to retrieve old data with reference to date or time. To test the practicality of the newly developed program, gene sequences of salivary mucin and amylase were examined to facilitate extraction of novel RNA tandem repeat element NASBA markers for human saliva forensic identification. Tandem repeats of non-coding portion with in the of human genome are highly polymorphic and considered best for forensic use. Currently, only 13 Short Tandem Repeat (STR) markers are available for forensic case work, which are not enough to establish a definitive link between the victim and suspects. Identification and validation of a new human body fluid tandem repeat markers is cruicial for achieving high through put results and to exonerate the innocent. / Access to thesis permanently restricted to Ball State community only / Department of Biology

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Biology

Made available in DSpace on 2015-07-13T12:10:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-01-23. Added 1 bitstream(s) on 2015-07-13T12:25:39Z : No. of bitstreams: 1 000825088_20160130.pdf: 143158 bytes, checksum: 1209a3e2f1bc3903da54429dc938e213 (MD5) Bitstreams deleted on 2016-02-01T10:15:52Z: 000825088_20160130.pdf,. Added 1 bitstream(s) on 2016-02-01T10:16:43Z : No. of bitstreams: 1 000825088.pdf: 567086 bytes, checksum: 73ca882633cceb536b683b63c338096b (MD5) / 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... / 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...

Nowadays intensive research is conducted for application of genetic polymorphisms for degraded samples analysis, identification and kinship determination. Another area of research in forensic genetics is biogeographical and phenotypic traits (eye, hair and skin color) determination. First part of presented work dealt with population study on the Czech popu- lation using Investigator DIPplex (QIAGEN, Germany) marker set containing 30 autosomal insertion-deletion polymorphisms. Power of Discrimination (PD), which is the probability of random selection of two persons with different genotypes, was 99.9999999999% for the whole marker set. This part of study concluded that ana- lyzed marker set is suitable as an additional marker panel for identification and kinship determination in the Czech Republic. Second part of the presented study was devoted to population research of Cen- tral Croatia using Mentype Argus X-8 kit (QIAGEN, Germany) containing 8 short tandem repeat polymorhisms located on X choromosomes (X-STR) divided into 4 linkage groups. PD for the whole kit reached 99.9999% and 99.99999999% for males and females, respectively. This kit could be used in Central Croatian population for kinship analysis and for identification as an additional marker panel. The next part of the presented study was the...

Philosophiae Doctor - PhD / This thesis has been placed under a long term embargo. Forensic and population genetic parameters were investigated in the Botswana population using autosomal and Y-chromosome short tandem repeat markers. AmpFlSTR Profiler plus markers were used to investigate the genetic diversity and forensic parameters in 773 individuals from Botswana from the reference database of the Botswana Police. The levels of polymorphism found using the AmpFlSTR Profiler Plus markers showed that the nine loci that make up the AmpFlSTR Profiler Plus can differentiate individuals for forensic casework in the Botswana population. AmpFlSTR Identifiler autosomal STR markers were used to investigate the population structure according to ethno-linguistics and geography 990 individuals from Botswana that serve as a reference database for the Botswana Police. Using pairwise genetic distances (Fst), analysis of molecular variance (AMOVA), factorial correspondence analysis (FCA), and the unsupervised Bayesian clustering method found in STRUCTURE and the landscape genetics software TESS, ethno-linguistics were found to have a greater influence on population structure than geography. The patterns of population structure found using these markers highlight the need for regional reference databases that include both ethnolinguistic and geographic location information. These markers have important potential for bio-anthropological studies as well as for forensic applications. The 17 Y-chromosomal short tandem repeats found in AmpFlSTR Y-filer and a highly discriminatory Y-STR genotyping system (the Y-STR 10-plex developed in the Forensics DNA Laboratory at the University of the Western Cape) were analysed in 249 unrelated male individuals from Botswana. Rst, multi-dimensional scaling (MDS) and AMOVA were used to investigate population differentiation in Botswana. The discrimination capacity (DC) was found to be higher using the Y-STR 10-plex as compared to the 17 markers in the Y-filer genotyping system. No geographic regional or ethnic differentiation was observed between the Northern and Southern regions of Botswana using both marker systems. Regional and ethnic variation can be useful in forensic working hypotheses. Cluster analysis using the highly discriminatory Y-STR 10-plex haplotypes may provide information about ancestry and haplogroup information. / National Research Foundation (NRF)

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-19T13:40:55Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese final.pdf: 4052323 bytes, checksum: 74297c119131b87a26908be1d213027a (MD5) / Made available in DSpace on 2016-07-19T13:40:55Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese final.pdf: 4052323 bytes, checksum: 74297c119131b87a26908be1d213027a (MD5) Previous issue date: 2015-12-01 / CAPEs / O uso de insetos visando responder aos quesitos levantados em investigações criminais ganhou espaço nas últimas décadas entre os pesquisadores e profissionais desta área, assim como a combinação de técnicas de genética forense para a obtenção de DNA humano a partir destes organismos, em especial dos dípteros necrófagos. Desse modo, neste estudo objetivou-se obter e testar um protocolo de identificação de DNA humano extraído a partir de larvas de dípteros coletadas em cadáveres no Instituto de Medicina Legal de Pernambuco Antonio Persivo Cunha (IMLAPC/PE). Inicialmente, espécimes imaturos foram coletados no IMLAPC/PE e criados em dieta a base de carne moída bovina para possibilitar a identificação da espécie mais abundante e frequente que se cria neste substrato. A espécie Chrysomya albiceps (Diptera: Calliphoridae) foi selecionada como modelo experimental. Grupos de larvas dessa espécie foram submetidos a uma dieta baseada em carne moída e sangue humano por 48 horas, dissecadas e submetidas a extração de DNA, utilizandose duas metodologias comumente adotadas pelos laboratórios de genética forense: Kit DNA IQ™ e Método Fenol-Clorofórmio. O DNA extraído foi quantificado através de Nanodrop® e Real-Time PCR 7500 com uso do Quantifiler® Duo DNA Quantification. Para amplificação do DNA foram usados os kits para STR (short tandem repeats): AmpFℓSTR® Identifiler® Plus PCR Kit, Argus X-12® Kit e PowerPlex® Fusion System kit. As amostras amplificadas foram analisadas por eletroforese capilar em ABI PRISM 3500, permitindo observar que, para os kits utilizados houve perfis íntegros e compatíveis com a amostra referência, a partir da extração com kit DNA IQ™ e/ou método Fenol- Clorofórmio. Além disso, foram testados quatro meios de armazenagem comumente utilizados em zoologia: etanol 70%, etanol 95%, formol 4% e via seca. Após 24 horas de armazenagem, as amostras foram submetidas aos processos de análise de DNA e o formol 4% apresentou os melhores perfis de DNA. O fato de haver perfis passíveis de comparação confirma a utilidade das larvas de dípteros usadas para este fim, as quais podem futuramente ser usadas para correlacionar perfis genéticos com uma cena criminal. O aprimoramento destas técnicas é necessário para que o uso das larvas de dípteros muscóides com emprego para a entomogenética tenha mais difusão entre os meios acadêmico e forense. / The use of insects for investigations has gained ground in recent decades among researchers and criminal professionals. Recently, the use of these animals has been combined with forensic genetics techniques for obtaining human DNA from these. Among the main focus groups for this technique are the carrion flies that have the host DNA extracted from intestinal contents. Because the visibility of this branch of forensic biology, this study aimed to obtain and test a protocol for identifying human DNA extracted from larvae of Diptera at the Instituto de Medicina Legal de Pernambuco Antonio Persivo Cunha (IMLAPC/PE). The species Chrysomya albiceps (Diptera: Calliphoridae) was selected as an experimental model. Groups of larvae of this species were subjected to diet ground meat and human blood for 48 hours, dissected and subjected to DNA extraction using two methods commonly used by forensic genetics laboratories: DNA IQ™ Kit and Method phenol-chloroform. The extracted DNA was quantified by Nanodrop® and Real-Time PCR 7500 with use of Quantifiler® Duo DNA Quantification. For DNA amplification kits for STR (short tandem repeats) were used: AmpFℓSTR® Identifiler® Plus PCR Kit, Argus X-12® Kit and PowerPlex® Fusion System kit. The amplified samples were analyzed by capillary electrophoresis in ABI PRISM 3500, allowing to observe for kits used there have upright profiles and compatible with the reference sample, from the IQ™ DNA extraction kit and/or phenol-chloroform method. In addition, four storage means commonly used in zoology were tested: 70% ethanol, 95% ethanol, 4% formaldehyde and dry. After 24 hours of storage, the samples were submitted to DNA analysis processes and the 4% formaldehyde DNA showed the best profile. The fact that there be comparable profiles confirms the usefulness of dipteran larvae used for this purpose, which can further be used to correlate genetic profiles and a criminal scene. The improvement of these techniques is required for the use of larvae dipterae with employment for the entomogenetics have more diffusion among academic and forensic means.

Orientadores: Eduardo Daruge Junior, Darcy de Oliveira Tosello / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-12T19:58:07Z (GMT). No. of bitstreams: 1 Santos_LeonardoSorianodeMello_M.pdf: 882529 bytes, checksum: a98503a69a38c35d2491b2e9cd210308 (MD5) Previous issue date: 2009 / Resumo: Alguns fatores relacionados ao estado e lugares que dentes humanos se encontram, nos que diz respeito a estes enquanto amostras com finalidade forense, ainda constituem desafio ao que tange o uso dos mesmos como material para obtenção de perfis genéticos de DNA. Este estudo visou comparar a extração de DNA feita a partir de dentes humanos com a extração por meios de amostras de sangue fixadas em papel FTA® utilizadas como grupo controle, de maneira a comparar os alelos mapeados e definir se os dentes constituem nestas circunstâncias, fonte viável de amostras para obtenção de perfis genéticos, comparando os protocolos. Dezoito participantes foram abordados e, aceitaram participar da pesquisa por meio de TCLE's, doaram voluntariamente amostras de sangue e os elementos dentários terceiros molares superiores direitos, estes indicados para exodontia por outros profissionais. Verificou-se que os dentes humanos constituíram fontes viáveis de acordo com a análise estatística realizada (Teste de Poisson), onde p<0,0001, entretanto quando comparado com o protocolo de extração de material genético através do sangue, deixa de ser viável devido ao número de passos necessários para a obtenção dos resultados. Ainda, 78,125% dos alelos possíveis de serem mapeados, o foram com sucesso / Abstract: Several factors related to how and where human teeth are found in forensic cases still a challenge to obtain genetic DNA profiles, as using theses elements as source for genetic material. This study aimed to compare the DNA extraction done through blood stains in FTA® paper cards, used as control group, and compare the mapped alleles from these to ones extracted from human teeth samples, as the simplicity of theses protocols when in comparison. Eighteen participants were convinced to join this study. Blood samples and superior right third molars (element 18) were donated. As result, teeth provided good sources of biologic sampling to obtain genetic profiles when analyzed by Poisson statistic analysis (p<0,0001), however, when compared to genetic material extraction protocol by blood, teeth analysis is no longer viable due to extensive laboratorial steps in order to gain the same results. Also 78,125% of the possible locci to be mapped and amplified were indeed / Mestrado / Odontologia Legal e Deontologia / Mestre em Biologia Buco-Dental

There is a transition nationwide on the analysis of forensic evidentiary stains containing biological material from traditional serology to Polymerase Chain Reaction (PCR) methodologies. The increased sensitivity of PCR, the limited number of alleles at each locus, and the necessity of producing unambiguous data for entry into the FBI's Combined DNA Index System make this study of extraction procedures of utmost importance. A "single tube" extraction procedure for blood stains collected onto FTA™ paper and a modified differential nonorganic extraction method from spermatozoa containing mixed stains were analyzed and compared. The extraction success was evaluated by amplification and typing of the amplified fragment length polymorphism, D1S80. These modifications of the nonorganic method utilized gave an improved separation of the spermatozoa-containing mixed stains.

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