The ability to confidently obtain deoxyribonucleic acid (DNA) profiles from a variety of sample types in a crime laboratory is very important. The first step in the analysis of DNA in a forensic crime laboratory is the extraction of the nucleic acid material from the rest of the cellular material contained in the sample. There are many different extraction methods available for use in the field of forensics but one that is reliable, cost effective, and easy to use is necessary. One of the methods that meet these requirements is a solid-phase extraction utilizing a silica membrane that binds the DNA in the presence of chaotropic salts. This solid-phase extraction using a silica membrane is ideal for automation and use with a bio-robot. One commonly used instrument is the BioRobot EZ1® system (Hilden, Germany) from Qiagen®. Automation using these robots was the first step in decreasing the time it takes to perform extraction as well as reduce the potential for contamination, but there are still opportunities to improve in both of these areas. In this study the Qiagen® Lyse&Spin Basket Kit was evaluated due to its potential to further decrease the time needed for extraction and the eliminate a step of the extraction process where contamination could be introduced. Laboratories around the country have reported problems with using Buffer G2 as an extraction buffer when used with samples such as blood on fabric. The reason for this is currently unknown, but in order to continue to confidently extract samples of this type, a change to diluted ATL buffer was suggested. The current extraction buffer, G2, used in the extraction protocol at the Kansas City Police Crime Laboratory was not yielding results in some situations such as blood on fabric. Switching to diluted ATL yielded the same quantity of DNA and the same quality of DNA profiles with mock casework samples. When diluted ATL was used with Quality Control (QC stain) samples it yielded significantly more DNA during extraction. When the Qiagen® Lyse&Spin Basket Kit was used during the extraction of whole blood on fabric (QC stain samples) they showed a significantly lower quantitation value than the tubes currently used in the Kansas City Police Department Crime Laboratory. The same results were obtained when the Qiagen® Lyse&Spin Basket Kit was used to extract 1:2 diluted saliva samples. When samples with lower quantities of DNA, such as 1:100 blood dilutions, 1:50 saliva dilutions, and mock casework samples, were examined the Qiagen® Lyse&Spin Basket Kit there were no significant differences seen when compared to the currently used baskets. When the quantitation data was analyzed for the QC stain samples extracted with the Qiagen® Lyse&Spin Basket Kit, abnormally high degradation index (DI) values were observed. It was determined that these high values did not affect the integrity of the sample. In order to determine the possible cause behind the poor performance of the Qiagen® Lyse&Spin Basket Kit when used to extract samples with higher starting amounts of DNA an experiment was designed to determine if genetic material was left on the cotton swatch in the spin basket. It was seen that DNA was left behind on the cotton swatches from both the Qiagen® Lyse&Spin Baskets and the currently used baskets. It appeared that more DNA was contained on the fabric used with the Qiagen® Lyse&Spin Basket, but further research is needed to determine if this difference is significant.

In 2010, Bright et al. created two person mixtures to determine how effective traditional differential extraction was in determining mixtures by examining mixture proportion variation by using the peak heights from each sample. This project aims to follow that method, however, in this case using a Temperature Controlled Differential Extraction (TCDE) to analyze post coital swabs in place of a traditional differential extraction. The project also aims to determine how efficient the separation of sperm cells from epithelial cells was by comparing the mixture proportion mean of male deoxyribonucleic acid (DNA) from an Acrosolv digest that did not undergo the TCDE to the proportion of male DNA from the TCDE. The amount of DNA remaining on a swab after undergoing the TCDE was also assessed as a material fraction. Many of the material fractions generated a mixture in their profiles and thus enough DNA to generate a male profile was remaining on the swab after the TCDE in almost all cases. The sperm fractions were mostly single source male profiles or profiles with the male DNA as a major contributor and the female DNA as a minor contributor.

Formalin-fixed, paraffin embedded (FFPE) tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research and clinical applications. However, FFPE DNA extraction presents unique challenges and requires lengthy incubation periods, which can be impractical for certain applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE generates a dense bubble cloud of acoustic cavitation capable of ablating tissue into an acellular lysate. FUSE treatment was applied to de-paraffinized porcine pancreas FFPE scrolls, followed by heated incubation for formaldehyde-induced DNA-protein crosslink reversal. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity ratios compared to conventional methods. DNA extracted via FUSE showed comparable fragmentation to conventional methods, and three out of four samples successfully amplified via PCR, indicating suitability for downstream analysis. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications. / Master of Science / Formalin-fixed, paraffin embedding (FFPE) has historically been the most popular method of biological tissue preservation, as it allows tissue to remain shelf stable for decades. As such, FFPE tissue is the most abundant, accessible, and versatile tissue sample type available for genetic research applications. Here, we propose the use of focused ultrasound extraction (FUSE) technology for improved DNA extraction from FFPE tissue. FUSE treatment applies rapid, focused ultrasound waves to tissue, resulting in the mechanical breakdown of cells and subsequent release of DNA. FUSE treatment was applied to pig pancreatic FFPE samples. When applied for 30 minutes, FUSE was found to successfully extract DNA from FFPE tissue as defined by increased DNA yield and improved purity compared to conventional methods. Three out of four DNA samples extracted via FUSE were successfully amplified, and DNA fragment lengths were comparable between FUSE and conventional methods, showing that FUSE did not fragment DNA beyond useful fragment lengths. These findings suggest that FUSE has the potential to increase the efficiency and effectiveness of DNA extraction from FFPE tissue. Further development and optimization of this protocol could develop a streamlined, easy to use extraction method that would simplify FFPE DNA extraction methods and address the primary time constraints which currently make FFPE DNA extraction time-consuming and impracticable for high-throughput applications.

<p>The broader aim of this work was to investigate the implementation of metagenomic library construction and sequencing-based approaches, as a basis for gene identification and functional characterization, from a novel thermophilic environment.</p>

The aim of the project was to develop a PCR method to detect HLA-B27 at the Immunology Department of St. James hospital in Dublin. The HLA-B27 gene is common among patients with ankylosing spondylitis (AS). Ninety percent of patients with AS have the HLA-B27 gene and it is therefore counted as a risk factor and could be used as part of the diagnosis. Twenty-two frozen blood samples from patients with AS or suspected AS were donated from the rheumatology department at St. James hospital. PCR is a well known and common technique, many hospital laboratories have a PCR machine and therefore PCR is a good choice for detection of the HLA-B27 gene. A multiplex PCR was developed where a PCR control, primers to the β-globin gene, was used in the same tube as the HLA-B27 primers, to secure that the PCR worked in every tube. Finally a blind test was performed to test the specificity of the PCR. The result shows that the specificity was 100%. Of all patient samples, sixteen was HLA-B27 positive and six were HLA-B27 negative. In addition, optimal conditions for the PCR and the way to extract DNA from frozen blood were successfully established. For future diagnosis, the described PCR can be used to detect the HLA-B27 gene in patients and it can be considered as a start for further development of a real-time PCR for detection of the HLA-B27.

<p>The broader aim of this work was to investigate the implementation of metagenomic library construction and sequencing-based approaches, as a basis for gene identification and functional characterization, from a novel thermophilic environment.</p>

Orientador: Lígia Souza Lima Silveira da Mota / Resumo: O Brasil é um país megadiverso detentor de grande parte da riqueza ecológica do mundo, porém seus biomas, importantes devido aos serviços ecossistêmicos, sofrem com a pressão antrópica. Assim, os grandes predadores, que possuem efeito regulatório no ecossistema, geram conflitos direto com o homem. O Puma concolor é um predador generalista, atuando no perfil trófico onde reside. Estudos de populações in situ são importantes na elaboração de planos de manejo e a utilização de amostras não invasivas nos permitem ter acesso a informações biológicas a baixo custo. O objetivo deste trabalho foi avaliar o uso de amostras não invasivas para contribuir com informações nos planos conservacionistas da região. Foram percorridas bordas de mata e áreas de transição da APA-Botucatu. As amostras de fezes encontradas foram georreferenciadas, coletadas, selecionadas, fotografadas, classificadas e seu DNA extraído por meio da técnica empregando fenol/clorofórmio. O DNA obtido foi quantificado em espectrofotômetro e sua qualidade avaliada em eletroforese. Trinta e cinco amostras foram classificadas quanto ao seu grau de degradação, submetidas à extração de DNA e seu produto avaliado. A presença de material genético nas fezes frescas ou pouco degradadas apresentaram maiores concentrações de DNA além de melhor qualidade quando comparadas com as mais degradadas. Estes resultados evidenciam que, o DNA obtido empregando metodologia de baixo custo, possui quantidade e qualidade suficientes para seu em... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2001Bitstream added on 2014-06-13T19:14:37Z : No. of bitstreams: 1 barea_ja_me_botfm.pdf: 626955 bytes, checksum: 85be35c50f78253f7b270023ffe7687e (MD5) / Este trabalho visou a comparação de 5 diferentes métodos de extração de DNA, a partir de amostras de materiais de arquivo (tecido incluído em parafina, lâmina de hemograma corada e não corada com Leishman, lâmina de mielograma, gotas de sangue em Guthrie card) e fontes escassas (células bucais, 1 e 3 bulbos capilares, 2 mL de urina), para avaliar a facilidade de aplicação dos mesmos e possibilidade de amplificação desse DNA pela técnica de PCR. Os métodos incluíram digestão por proteinase K, seguida e não seguida por purificação com fenol/clorofórmio, utilização de Chelex 100 Ò, utilização de InstaGeneÒ e fervura em água estéril. O DNA obtido, foi testado por PCR, para a amplificação de três fragmentos gênicos: de Brainderived neutrophic factor (764 pb), de Fator V Leiden (220 pb) e de Abelson (106 pb), sendo que, a amplificação para o primeiro, eliminava a necessidade dos demais. Conforme o tamanho do fragmento gênico estudado, a fonte potencial de DNA e o método de extração utilizado, os resultados caracterizaram o melhor caminho para padronização dos procedimentos técnicos a serem incluídos e apresentados no manual de Procedimentos Operacionais Padrão do Laboratório de Biologia Molecular do Hemocentro – HC – UNESP – Botucatu. / The present work aimed to compare five different methods of DNA extraction of archieved materials samples (paraffin-embedded tissues, periferic blood smear – stained or non-stained with Leshman, aspired bone marrow smears and blood guts in Guthrie card) and rare sources (oral cells, 1 and 3 capilar bulbs, 2 mL urine), to avaliate the aplication facility and the amplification possibility one for PCR. The methods included proteinase K digestion – followed or non by phenol/chloroform purification, Chelex 100Ò (BioRad), InstaGeneÒ (BioRad) and boilling in sterile water. The DNA obteined, was tested for amplification of 3 genic fragments: from Brainderived neutrophic factor gene (764 bp), Factor V Leiden gene (220 bp) and Abelson gene (106 bp). According to the genic fragment lenght studed, the DNA potential source and the extraction method used, the results characterized better guidelines for padronization of the techniques procedures for to Good Manufacturing Practices from Molecular Biology Laboratory from Blood Center – Medicine School – UNESP - Botucatu .

Philosophiae Doctor - PhD / Metagenomic sequencing of environmental samples provide direct access to genomic information of organisms within the respective environments. This sequence information represents a significant resource for the identification and subsequent characterization of potentially novel genes, or known genes with acquired novel characteristics. Within this context, the thermophilic environments are of particular interest due to its potential for deriving novel thermostable enzymes with biotechnological and industrial applications. In this work metagenomic library construction, random sequencing and sequence analysis strategies were employed to enhance identification and characterisation of potentially novel genes, from a thermophilic soil sample. High molecular weight metagenomic DNA was extracted from two Chinese hydrothermal soil samples. This was used as source material for the construction of four genomic DNA libraries. The combined libraries were estimated to contain in the order of 1.3 million genes, which provides a rich resource for gene identification. Approximately 70 kbp of sequence data was generated from one of the libraries as a resource for sequence-based analysis. Initial BLAST analysis predicted the presence of 53 ORFs/partial ORFs. The BLAST similarity scores for the investigated ORFs were sufficiently high (>40%) to infer homology with database proteins while also being indicative of novel sequence variants of these database matches. In an attempt to enhance the potential for deriving more full length ORFs a novel strategy, based on WGA technology, was employed. This resulted in the recovery of the near complete sequence of partial ORF5, directly from the WGA DNA of the environmental sample. While the full length ORF5 could not be recovered, the feasibility of this novel approach, for enhanced metagenomic sequence recovery was proved in principle. The implementation of multiple insilico strategies resulted in the identification of two ORFs, classified as homologs of the DUF29 and Usp protein families respectively. The functional inference obtained from the integrated in-silico predictions was furthermore highly suggestive of a putative nucleotide binding/interaction role for both ORFs. A putative novel DNA polymerase gene (denoted TC11pol) was identified from the sequence data. Expression and characterization of the full length TC11pol did however not result in detectable polymerase activity. The implementation of a homology modeling approach proved succesfull for deriving a structural model of the polymerase that was used for: (i) deriving functional inferences of the potential activities of the polymerase and (ii) deriving a 5’ exonuclease deletion mutant for functional analysis. Expression and subsequent functional characterization of the putative 5’exo- TC11pol mutant resulted in detectable polymerase and 3’-5’ exonuclease activity at 37 and 45 oC, following a heat denaturation step at 55 oC for 1 hour. It was, therefore concluded that the putative 5’exo- TC11pol mutant was functionally equivalent to the Klenow fragment of E. coli, while exhibiting increased thermostability. / South Africa

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