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1
Rifaat, Rasekh. "Multifractal analysis of DNA." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0007/MQ32231.pdf.
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Stephens, Nathan W. "A comparison of genetic microarray analyses : a mixed models approach versus the significance analysis of microarrays /." Diss., CLICK HERE for online access, 2006. http://contentdm.lib.byu.edu/ETD/image/etd1604.pdf.
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McClelland, Robyn L. (Robyn Leagh). "Statistical analysis of DNA profiles." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=68215.
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DNA profiles have become an extremely important tool in forensic investigations, and a match between a suspect and a crime scene specimen is highly incriminating. Presentation of this evidence in court, however, requires a statistical interpretation, one which reflects the uncertainty in the results due to measurement imprecision and sampling variability. No consensus has been reached about how to quantify this uncertainty, and the literature to date is lacking an objective review of possible methods.This thesis provides a survey of approaches to statistical analysis of DNA profile data currently in use, as well as proposed methods which seem promising. A comparison of frequentist and Bayesian approaches is made, as well as a careful examination of the assumptions required for each method.
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O'Donoghue, Kerry. "Chemical analysis of ancient DNA." Thesis, University of Manchester, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488296.
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Akman, Kemal. "Bioinformatics of DNA Methylation analysis." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-182873.
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Hastings, Patsy-Ann Susan. "MITOCHONDRIAL DNA ANALYSIS BY PYROSEQUENCING." Master's thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4447.
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Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality. The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.M.S.
Department of Chemistry
Arts and Sciences
Chemistry
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Wang, Meng. "Mutational analysis of DNA deaminases." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611829.
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Salman, Abbas Ali Abulwohab. "Miniaturised system for DNA analysis." Thesis, Teesside University, 2013. http://hdl.handle.net/10149/316214.
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The growing markets for analytical techniques in areas such as pathogen detection, clinical analysis, forensic investigation, environmental analysis and food analysis require the development of devices with simultaneous high performance, speed, simplicity and low cost. Analysis of deoxyribonucleic acid (DNA) has been enhanced by use of the polymerase chain reaction (PCR) technique, which is now a widely used tool for in vitro amplification of nucleic acids. In this work, a miniaturised PCR system comprising a microfluidic PCR chip, novel heating method and fluorescence detection unit was developed. PCR chip with reactants were shunted along three temperature zones in a fine polycarbonate chip. The polycarbonate PCR chip was fabricated using milling and thermal fusion binding for sealing of the cover. Thermal-cycling within the microfluidic chip was achieved by programmable shunting of the chip between three double side temperature zones with different temperatures to accomplish the denaturation, annealing and elongation steps necessary for PCR amplification. This thermal-cycling model potentially improves PCR efficacy because it increases the ramping rates for heating and cooling the PCR mixture. The detection unit comprises a photo-detector and Light Emitting Diode (LED) as the source of excitation. The detection limit of the system was determined on the PCR chip using Fluorescein isothiocyanate (FITC) as a fluorophore dye. The detection limit achieved was 7.8 pg ml-1 or (19.7 pmol) of FITC. The chromosomal DNA used in this work was extracted from non-pathogenic K-12 subtype of Escherichia coli (E. coli). The investigations showed that the system was capable of performing PCR amplification with different annealing temperature ranging from 54 to 68 °C, targeting three different sizes of PCR products of 250, 552 and 1500 bp. The prototype thermal-cycler and PCR chip were used successfully to amplify the three sizes and the results were compared with same fragments amplified on a conventional PCR .thermal-cycler machine. The method used for comparison was gel electrophoresis. In addition, a fluorescence detection system was employed for detecting of PCR products using SYBR Green I fluorescent dye. The whole system allows for developments of low cost, easy to use and portable instruments.
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Poli, Elena. "DNA METHYLATION ANALYSIS IN RHABDOMYOSARCOMA." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424380.
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Rhabdomyosarcoma (RMS) is a highly aggressive pediatric soft-tissue sarcoma. It is mainly classified into two major subtypes characterized by alveolar (ARMS) and embryonal (ERMS) histologies. ARMS are characterized by a more aggressive behavior with a higher tendency to present metastasis at diagnosis and to relapse after treatment. Approximately 80% of ARMS harbour the reciprocal chromosomal translocation t(2;13)(q35;q14) and, less commonly, the variant translocation t(1;13)(p36;q14), in which PAX3 and FOXO1, or PAX7 and FOXO1 genes, respectively, are juxtaposed. Unfortunately, no such specific genetic aberrations are known in ERMS, and myogenic factors as myogenin and MyoD1 are the only diagnostic indicators that can be used. Despite aggressive multimodal therapies, the prognosis of high-risk RMS patients has not been improved, with a 5-year overall survival rate less than 20-30%, which prompts a need for new therapeutic strategies. In the last decade many scientific studies have demonstrated that gene expression signature distinguishes PAX3-FOXO1 positive RMS from PAX3-FOXO1 negative, but the reasons of the different expression are still unknown. Aberrant DNA methylation patterning is a hallmark of cancer and could be responsible for the different gene expression of RMS tumor subtypes.
We performed genome-wide methylation profile by microarray experiments followed by Reduced-Representation Bisulfite Sequencing (RRBS).
Microarray analysis demonstrated a different methylation profile between PAX3-FOXO1 positive and negative RMS, besides among metastatic and non-metastatic RMS. We confirmed HOXC11 as one of the gene differentially methylated between PAX3-FOXO1 positive and negative RMS cell lines using in vitro demethylating agents and bisulfite sequencing. Unfortunately, we did not validate the result in the cohort of RMS biopsies. Moreover, we performed another analysis on microarray data comparing metastatic vs non-metastatic RMS. We found an elevated numbers of differentially methylated regions (DMRs) and many of them map to promoter regions of genes implicated in tumors development. In particular we found DMRs linked to clustered protocadherins, known as tumor suppressor genes. We confirmed a different expression pattern of PCDHA4, as well as a different methylation level of its promotorial region, comparing metastatic and non-metastatic RMS samples. Nevertheless, the methylation status and the expression level of PCDHA4 did not have significant correlation with clinical features and are not a predictor of poor prognosis in RMS.
Then, we performed an RRBS sequencing, in order to validate data obtained with microarray platforms. We observed a very low concordance between the two approaches, probably caused by a low quality DNA used in microarray experiments. The RRBS sequencing had demonstrated again that PAX3-FOXO1 positive and negative RMS have a different methylation pattern. Moreover, we demonstrated that GADD45G and NELL1, already described as tumor suppressors in other cancers and often downregulated by methylation processes, had also an involvement in RMS biology. Our experiments confirmed an epigenetic regulation by DNA methylation for GADD45G and NELL1 and that their expression were correlated to RMS histology, presence of fusion status and IRS group staging. Furthermore, GADD45G and NELL1 expression levels affect the progression free survival of RMS patients suggesting their association with a poor prognosis.
In conclusion, we demonstrated that GADD45G and NELL1 could be novel potential biomarkers in RMS and we evidenced that the DNA methylation pattern in RMS could be interesting for new therapeutic strategies.
We hope that our efforts could contribute to a better molecular classification of RMS tumors and to the identification of new targets for improving standard therapy.Il rabdomiosarcoma (RMS) è una sarcoma pediatrico dei tessuti molli altamente aggressivo. Viene classificato principalmente in due sottotipi, caratterizzati da istologia alveolare (RMSA) o embrionale (RMSE). Nei RMSA si osserva un comportamento più aggressivo e una maggiore tendenza a presentare metastasi alla diagnosi e alla ricaduta dopo trattamento. Circa l'80% dei RMSA presentano la traslocazione cromosomica reciproca t(2; 13) (q35; q14) e, meno comunemente, la variante t(1; 13) (p36; q14), in cui i geni PAX3 e FOXO1, o PAX7 e FOXO1, rispettivamente, sono giustapposti. Purtroppo, non si conoscono aberrazioni genetiche specifiche nei RMSE e i fattori miogenici, come miogenina e MyoD1, sono gli unici indicatori diagnostici che possono essere utilizzati. Nonostante l’applicazione di terapie aggressive multimodali, la prognosi dei pazienti affetti da RMS, della categoria alto rischio, non è migliorata, con un tasso di sopravvivenza a 5 anni inferiore al 20-30%. Questo dato indica la necessità di sviluppare nuove strategie terapeutiche. Nell’ultimo decennio molti studi scientifici hanno dimostrato che in base al profilo di espressione genica è possibile distinguere RMS PAX3-FOXO1-positivi e PAX3-FOXO1-negativi, ma le ragioni di questa diversa espressione sono ancora sconosciute. L’anomala metilazione del DNA è un indicatore di neoplasia e potrebbe essere la causa responsabile della diversa espressione genica dei due sottotipi di tumore. In questo studio, per mezzo di esperimenti di microarray, abbiamo realizzato un’analisi dello stato di metilazione del DNA su tutto il genoma, proseguendo poi con esperimenti di sequenziamento sfruttando la tecnica Reduced-Representation Bisulfite Sequencing (RRBS). L’analisi dei risultati ottenuti con gli esperimenti di microarray ha dimostrato, non solo un profilo di metilazione diverso tra i RMS PAX3-FOXO1-positivi e negativi, ma anche tra i RMS metastatici e non metastatici. Abbiamo confermato che il gene HOXC11 risulta essere differenzialmente metilato tra linee cellulari di RMS PAX3-FOXO1-positive e negative, sfruttando trattamenti con agenti demetilanti in vitro e sequenziamento del DNA dopo conversione con bisolfito; purtroppo, non abbiamo confermato il risultato nella coorte di biopsie di RMS. Inoltre, abbiamo effettuato un'ulteriore analisi sui dati di microarray confrontando i RMS metastatici con i non metastatici. Abbiamo trovato un elevato numero di regioni differenzialmente metilate (DMR) e molte di queste sono risultate coincidere con le regioni promotoriali di geni implicati nello sviluppo di tumori; in particolare, abbiamo trovato DMR connesse alla famiglia delle clustered protocaderine, note come geni soppressori di tumore. Abbiamo poi confermato un diverso profilo di espressione del gene PCDHA4, così come un diverso stato di metilazione a livello della sua regione promotoriale, confrontando campioni di RMS metastatici e non metastatici. Tuttavia, lo stato di metilazione e il livello di espressione di PCDHA4 non hanno dimostrato una correlazione significativa con le caratteristiche cliniche del RMS. Il gene PCDHA4 non risulta quindi essere un predittore prognostico nel RMS. Successivamente, abbiamo effettuato un sequenziamento RRBS, al fine di validare i dati ottenuti con le piattaforme dei microarray. Ne è risultata una bassa concordanza tra i due approcci, probabilmente a causa della bassa qualità del DNA utilizzato negli esperimenti di microarray. Il sequenziamento RRBS ha dimostrato ancora una volta che i RMS PAX3-FOXO1-positivi hanno un profilo di metilazione diverso dai RMS PAX3-FOXO1-negativi. Inoltre, abbiamo dimostrato che GADD45G e NELL1, già descritti come soppressori tumorali in altri tipi di tumore e spesso regolati in maniera negativa da processi di metilazione, sono anche coinvolti nella biologia del RMS. Con i nostri esperimenti abbiamo confermato una regolazione epigenetica, mediata dalla metilazione del DNA ,per i geni GADD45G e NELL1, e come la loro espressione sia correlata alla istologia del RMS, alla presenza dei geni di fusione e alla stadiazione in gruppi IRS. Inoltre, abbiamo dimostrato che i livelli di espressione di GADD45G e NELL1 influenzano la sopravvivenza libera da progressione di malattia nei pazienti affetti da RMS, suggerendo la loro associazione con una prognosi sfavorevole. In conclusione, il nostro lavoro ha dimostrato che GADD45G e NELL1 potrebbero essere nuovi potenziali biomarcatori nel RMS, evidenziando come il profilo di metilazione del DNA nel RMS potrebbe favorire lo sviluppo di nuove strategie terapeutiche. Ci auguriamo che i nostri sforzi possano contribuire ad una migliore classificazione molecolare dei tumori nei pazienti affetti da RMS e alla identificazione di nuovi bersagli farmacologici per una terapia più mirata.
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Zhang, Jianhua. "Restriction fragment length polymorphism analysis of chloroplast DNA, mitochondrial DNA, and ribosomal DNA in turfgrasses." Diss., This resource online, 1994. http://scholar.lib.vt.edu/theses/available/etd-06062008-170748/.
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Patel, Yogen. "DNA methylation analysis of Alzheimer's disease." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/dna-methylation-analysis-of-alzheimers-disease(f66ad885-3fdd-4c12-a73a-921cc31ccac2).html.
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There is evidence for a role for epigenetic mechanisms in Alzheimer's disease (AD), the most common age-dependent neurodegenerative disorder. The most studied epigenetic mark DNA methylation -the addition of a methyl group to cytosines located in CpG dinucleotides (5mC) - is known to change with aging and may reflect subtle changes in gene expression. Recently a second type of modified cytosine - a hydroxylated and methylated form (5hmC) - has been detected in the brain and maybe linked to the regulation of gene expression. Case-control differences in post-mortem brain DNA methylation were sought by examining both global DNA methylation and DNA methylation of two candidate genes relating to AD risk factors. Simultaneous assessment of 5mC and 5hmC methylation at a global level indicate hypomethylation of 5mC and hypermethylation of 5hmC in AD brain relative to controls, consistent with the notion that 5hmC serves as an intermediary form for demethylation of 5mC. Age was separately associated with a decrease in LINEl methylation and an increase in 5hmC methylation. The comorbidity of depression in AD was explored by assessing the methylation status of the serotonin transporter (SERT) gene promoter across several brain areas and showed tentative associations of disease with SERT CpG methylation. These measurable differences are very small and unlikely to represent any biological plausibility. In a subset of AD patients with additional clinical and behavioural measures there was no effect of SERT 5HTTLPR genotype on DNA methylation. The hypothesis that amyloid- deposition in brain is a consequence of amyloid precursor protein (APP) gene over-expression was examined by measuring DNA methylation across the APP gene region. AD status associates with methylation levels of several CpG sites within the 5' region CGI shore and exon 5 of the APP gene. However there are no co-occurring separate associations of total APP protein levels at these CpG sites. This study demonstrates the utility of the Fluidigm microfluidics platform to generate highly parallel bisulphite sequencing/base pair resolution CpG data.
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Gomaa, R. "Analysis of mitochondrial DNA variation in the Egyptian population and its implications for forensic DNA analysis." Thesis, Nottingham Trent University, 2010. http://irep.ntu.ac.uk/id/eprint/357/.
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The genetic sequence of human mitochondrial DNA (mtDNA) is of particular interest to forensic investigations involving human identification, as well as population genetics. The current mtDNA database is lacking sufficient representatives of African mitochondrial DNA sequences compared to European and Asian sequences. The present study was concerned with the analysis of mtDNA in the Egyptian population. FTA cards were used for blood sample collection, storage, and shipment and DNA extraction. An optimised laboratory protocol for rapid PCR amplification of the mitochondrial hypervariable regions was developed. A database of 261 mitochondrial hypervariable region I (HVI) sequences and 78 hypervariable region II (HVII) sequences was established from 261 adult Egyptians. A total of 113 polymorphic sites were reported in the HVI region (nt16024-16365) which identified a total of 187 different haplotypes, of which 151 were unique to single individuals. The most commonly observed HVI haplotype was identical to the Cambridge Reference Sequence (CRS). Analysis of 78 HVII sequences (nt73-340) revealed a total of 42 polymorphic sites that identified 62 different haplotypes, of which 51 were unique to single individuals. Sites that showed the highest variability in the HVI and HVII regions agreed with the previously reported mutational hotspots. Combination of the HVI and HVII data resulted in identification of 207 different mitochondrial haplotypes, of which 183 (~88%) were unique to single individuals. Such a large number of unique mitochondrial haplotypes indicates a high diversity of mtDNA in the Egyptian population, which has a direct impact on forensic applications, since the significance of a match between an evidence sample and a reference sample depends on the population frequency of a profile. The random match probabilities in the HVI and HVII datasets were 2.34% and 3.22%, respectively. Combination of the two datasets reduced the random match probability to 1.28%. The genetic diversities in the HVI and HVII sequences were estimated to be 0.9804 and 0.9803, respectively, whereas, the genetic diversity in the combined dataset was 0.9911. A new strategy was developed to facilitate Restriction Fragment Length Polymorphism (RFLP) analysis of the whole mitochondrial genome for the purpose of haplogroup assignment. All the Egyptian individuals were assigned to well known mitochondrial haplogroups and the frequency distribution of different lineages was estimated. A mixed maternal ancestry of the Egyptian population was reported via detection of a mixture of mitochondrial DNA lineages with varying racial backgrounds. The most frequently reported mitochondrial lineages were haplogroup T (13.8%) followed by haplogroups L3 (12.6%), H (12.3%), U (~10%) and M (8.4%). The genetic relationship between Egypt and its neighbours was revealed, and it was found that present day Egyptians were the closest African population to the Middle East and Europe, with an overall 62.5% European, 25% African, and 12.5% Asian mitochondrial lineages. The data presented here will enrich the limited Egyptian mitochondrial DNA reference data currently available for forensic applications.
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Hare, Brian K. Dinakarpandian Deendayal. "Feature selection in DNA microarray analysis." Diss., UMK access, 2004.
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Thesis (M.S.)--School of Computing and Engineering. University of Missouri--Kansas City, 2004."A thesis in computer science." Typescript. Advisor: D. Dinakarpandian. Vita. Title from "catalog record" of the print edition Description based on contents viewed Feb. 24, 2006. Includes bibliographical references (leaves 81-86 ). Online version of the print edition.
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Nilsson, Martina. "Mitochondrial DNA in Sensitive Forensic Analysis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7458.
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Dahl, Fredrik. "Selector Technology : For Multiplex DNA Analysis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5921.
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Shah, Jayna J. "Microfluidic devices for forensic DNA analysis." Fairfax, VA : George Mason University, 2007. http://hdl.handle.net/1920/2878.
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Thesis (Ph. D.)--George Mason University, 2007.Title from PDF t.p. (viewed Jan. 22, 2008). Thesis director: Rao V. Mulpuri. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Electrical and Computer Engineering. Vita: p. 159. Includes bibliographical references (p. 145-158). Also available in print.
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Brandt, Regine, Robert Grützmann, Andrea Bauer, Ralf Jesenofsky, Jörg Ringel, Matthias Löhr, Christian Pilarsky, and Jörg D. Hoheisel. "DNA microarray analysis of pancreatic malignancies." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-136556.
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Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignanciesDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Meade, Andrew Paul. "The computational analysis of DNA sequences." Thesis, University of Reading, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412195.
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Murry, Robert Lester. "Continuum electrostatic analysis of DNA bending." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/38837.
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Ahnesorg, Peter. "Molecular genetic analysis of DNA repair." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614127.
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Pechstedt, Katrin. "A nanotechnology approach to DNA analysis." Thesis, University of Southampton, 2011. https://eprints.soton.ac.uk/340250/.
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This thesis describes the investigation of quantum dots and nano-structured metallic films for use in single genomic DNA analysis. The fluorescence of continuously illuminated core/shell CdSe/ZnS quantum dots (QDs) under various atmospheric conditions is investigated experimentally. Initial enhancement in fluorescence intensity is observed followed by degradation; both are highly dependent on the atmospheric conditions. Following a series of studies theories are put forward to explain these observations. Solution mixtures of DNA strands and QDs are imaged with Atomic Force Microscopy and Fluorescence Microscopy to investigate the binding properties of DNA strands with QDs. Gold nanovoids are fabricated for use as a substrate to provide localised enhanced fluorescence intensity of fluorescently labelled regions of DNA strands stretched and located over nanovoids as a result of resonant coupling with localised surface plasmon polariton modes. The energy and electric field distribution of localised surface plasmon polaritons is considered for various void geometries for use with fluorescently labelled DNA. The experimental fluorescence intensity profile along fluorescently labelled DNA strands stretched over glass, electrochemically grown gold and gold nanovoid substrates is compared and the fluorescence lifetime is measured. Short fluorescence lifetimes and increased intensities over gold nanovoids and a constant lifetime over glass are observed. The results of these biophysical studies are discussed with a view for application as methods for distinguishing different DNA sequences on the nanoscale.
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Brandt, Regine, Robert Grützmann, Andrea Bauer, Ralf Jesenofsky, Jörg Ringel, Matthias Löhr, Christian Pilarsky, and Jörg D. Hoheisel. "DNA microarray analysis of pancreatic malignancies." Karger, 2004. https://tud.qucosa.de/id/qucosa%3A27711.
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Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment are needed. Recent advances in human genome research and high-throughput molecular technologies make it possible to cope with the molecular complexity of malignant tumors. With DNA array technology, mRNA expression levels of thousand of genes can be measured simultaneously in a single assay. As several studies using microarrays in PDAC have already been published, this review attempts to compare the published data and therefore to validate the results. In addition, the applied techniques are discussed in the context of pancreatic malignancies.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Jones, Nathan Jones. "Single Molecule Analysis of DNA Interactions." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1511959163350735.
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Watt, Kathryn Elizabeth. "Decontamination techniques in ancient DNA analysis /." Burnaby B.C. : Simon Fraser University, 2005. http://ir.lib.sfu.ca/handle/1892/2446.
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Molak, Martyna. "Evolutionary Analysis of Ancient DNA Sequences." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/11674.
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Studies of genetic data from old specimens – ancient DNA – offer a unique possibility to explore evolutionary history. Ancient DNA allows examination of past populations, extinct prehistoric species, and even large-scale analyses of past ecosystems. Since the beginnings of ancient DNA research in the mid 1980s, the field has undergone major technological and methodological transitions. However, investigations of the theory and methods used to analyse ancient DNA sequences have not kept up with the rapid pace of data generation. This thesis investigates a number of issues associated with the evolutionary analysis of ancient DNA. First, I built a model of post-mortem DNA degradation and examined its potential effects on the resulting sequence data (Chapter 2). Second, I conducted a large-scale study of time-dependent rates in order to look into their causes, characteristics, and ubiquity (Chapter 3). Third, I explored the impact of incorporating uncertainties associated with sample age into phylogenetic analysis (Chapters 4 and 5). Fourth, I evaluated the performance of Bayesian skyline plots in detecting recent population bottlenecks (Chapter 6). Finally, I combined several of these developments in a biogeographical analysis of the extinct cave bear (Chapter 7). My thesis shows that many of the common practices in ancient DNA studies, such as employing sequence-authentication criteria or ignoring age uncertainty in phylogenetic analyses, appear to be effective and to produce reliable results. However, there are areas in which more caution is needed when interpreting the results of DNA analyses. These include estimations of evolutionary rates, which are highly sensitive to the calibration points that are used; and the poor performance of Bayesian skyline reconstructions of recent population-size changes. The results of my studies improve our understanding of ancient DNA research and will serve as a useful guide for future evolutionary analyses of ancient DNA sequences.
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Bowler, Frank Ray. "Reading DNA with PNA : a dynamic chemical approach to DNA sequence analysis." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5270.
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Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) constitute important sources of genetic variation which provide insight into disease aetiology and idiosyncratic differences in drug response. The analysis of such genetic variation relies upon the generation of allele-specific products, typically by enzymatic extension or the hybridization of allele-specific DNA probes. Herein, a distinct enzyme-free, dynamic chemistry-based method of producing allele-specific products for genotyping was developed. The approach was initially demonstrated in model systems using synthetic DNA, which was used as a template in a base-filling reductive amination reaction on a PNA backbone. The templated dynamic reaction between a free secondary amine at a ‘blank’ position on the PNA strand and four aldehyde-modified nucleobases drove selective formation of the ‘correct’ iminium intermediate according to Watson-Crick base-pairing rules. In a blind trial, the method was extended to genotype twelve cystic fibrosis patients for two mutations (one SNP and one indel) linked to this disease. Enzyme-free dynamic chemistry thus permitted successful genotyping in both singleplex and duplex formats, demonstrating the application of dynamic chemistry as a distinct method of allelediscrimination with certain advantages over those reported previously. The application of this method as a tool for the discovery of non-natural nucleobases with improved properties for antisense and genotyping applications was also investigated. Furthermore, progress was made towards the use of dynamic chemistry as a means of full nucleic acid sequence analysis, through the templated sequence-selective extension of PNA probes by reductive amination.
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Berge, Torunn. "Structural analysis of DNA and DNA-protein complexes using atomic force microscopy." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621339.
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Wang, Suyue. "Characterization of a Human 28S Ribosomal RNA Retropseudogene and Other Repetitive DNA Sequence Elements Isolated from a Human X Chromosome-Specific Library." Thesis, University of North Texas, 1994. https://digital.library.unt.edu/ark:/67531/metadc278083/.
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Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.
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Savill, Jane Marie. "Analysis of DNA damage sensing and DNA recombination using high-throughput functional genomics." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608872.
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Easthon, Lindsey. "Conformational analysis of E. coli DnaT and the complex with PriA N-terminal domain." University of Toledo / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1271434912.
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Thomas, Adam David. "Mechanistic investigations of DNA reactive carcinogens at low dose, through analysis of DNA adducts, mutations and DNA repair." Thesis, Swansea University, 2012. https://cronfa.swan.ac.uk/Record/cronfa43089.
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Genetic toxicology assesses the genotoxic potential of chemicals in consumer products, pharmaceuticals and from agricultural and industrial processes. Such assessment is integral in hazard identification and risk assessment to prevent unnecessary human exposure and limit cancer risk. Human risk assessments for genotoxic alkylating agents were based upon linear dose-response models where genotoxicity accrues proportionally with dose. Evidence is accumulating to support a non-linear dose-response at low doses of ethyl methanesulfonate (EMS), a model alkylating agent. For acceptance of non-linear dose responses, a strong explanatory mechanism of action needs to be elucidated. In the following work, low dose mutagenic effects of methyl nitorosurea (MNU), the most potent alkylating agent, have been examined in AHH-1 human lymphoblastoid cells using the HPRT assay. An increase in mutant frequency was not observed until 0.01pg/ml MNU (LOGEL, Lowest Observed Genotoxic Effect Level) with a No-Observed Genotoxic Effect Level (NOGEL) at 0.0075pg/ml MNU. Of interest, is the apparent hormesis induced at 0.0025pg/ml MNU. The principle adduct responsible for MNU mutagenesis is 0 6Methylguanine (06MeG) that miscodes during replication and becomes fixed as GC→AT transitions. Accordingly, the non-linear increase in mutant frequency is accompanied by a non-linear increase in GC→AT transitions. Furthermore, evidence is provided that implicates methlyguanine methyltransferase (MGMT) in protecting DNA from MNU induced mutagenesis by repairing 0 6MeG at low doses, thereby creating the NOGEL. AHH-1 cells treated with 0 6Benzylguanine (06BG), to inactivate MGMT, were hypersensitive to low dose MNU mutagenesis. At 0.0075pg/ml MNU, there was a three-fold increase in mutant frequency and an increase in proportion of GC-^AT transitions, from 28% to 48% in MGMT inactivated cells. This thesis presents a non-linear dose-response for MNU with a strong biological mechanism of action involving DNA repair.
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Ola, Ayodele Oluronke. "A functional analysis of proliferating cell nuclear antigen (PCNA)." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391441.
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Lee, Kyeong Eun. "Bayesian models for DNA microarray data analysis." Diss., Texas A&M University, 2005. http://hdl.handle.net/1969.1/2465.
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Selection of signi?cant genes via expression patterns is important in a microarray problem. Owing to small sample size and large number of variables (genes), the selection process can be unstable. This research proposes a hierarchical Bayesian model for gene (variable) selection. We employ latent variables in a regression setting and use a Bayesian mixture prior to perform the variable selection. Due to the binary nature of the data, the posterior distributions of the parameters are not in explicit form, and we need to use a combination of truncated sampling and Markov Chain Monte Carlo (MCMC) based computation techniques to simulate the posterior distributions. The Bayesian model is ?exible enough to identify the signi?cant genes as well as to perform future predictions. The method is applied to cancer classi?cation via cDNA microarrays. In particular, the genes BRCA1 and BRCA2 are associated with a hereditary disposition to breast cancer, and the method is used to identify the set of signi?cant genes to classify BRCA1 and others. Microarray data can also be applied to survival models. We address the issue of how to reduce the dimension in building model by selecting signi?cant genes as well as assessing the estimated survival curves. Additionally, we consider the wellknown Weibull regression and semiparametric proportional hazards (PH) models for survival analysis. With microarray data, we need to consider the case where the number of covariates p exceeds the number of samples n. Speci?cally, for a given vector of response values, which are times to event (death or censored times) and p gene expressions (covariates), we address the issue of how to reduce the dimension by selecting the responsible genes, which are controlling the survival time. This approach enables us to estimate the survival curve when n << p. In our approach, rather than ?xing the number of selected genes, we will assign a prior distribution to this number. The approach creates additional ?exibility by allowing the imposition of constraints, such as bounding the dimension via a prior, which in e?ect works as a penalty. To implement our methodology, we use a Markov Chain Monte Carlo (MCMC) method. We demonstrate the use of the methodology with (a) di?use large B??cell lymphoma (DLBCL) complementary DNA (cDNA) data and (b) Breast Carcinoma data. Lastly, we propose a mixture of Dirichlet process models using discrete wavelet transform for a curve clustering. In order to characterize these time??course gene expresssions, we consider them as trajectory functions of time and gene??speci?c parameters and obtain their wavelet coe?cients by a discrete wavelet transform. We then build cluster curves using a mixture of Dirichlet process priors.
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Seldeen, Kenneth Ladd. "Biophysical Analysis of the AP1-DNA Interaction." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/259.
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Jun and Fos are components of the AP1 family of transcription factors that bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. The specific protein-DNA interactions are driven by the binding of basic zipper (bZIP) domains of Jun and Fos to TPA response element (TRE) and cAMP response element (CRE) within the promoters of target genes. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry in conjunction with molecular modeling and semi-empirical analysis, I characterize AP1-DNA interactions in thermodynamic and structural terms. My data show that the binding of bZIP domains of Jun-Fos heterodimer to TRE and CRE are under enthalpic control accompanied by entropic penalty at physiological temperatures. This is in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. A larger than expected heat capacity change suggests that the basic regions within the bZIP domains are unstructured in the absence of DNA and interact in a coupled folding and binding manner. Further analysis demonstrates that Jun-Fos heterodimer can tolerate single nucleotide variants of the TRE consensus sequence and binds in the biologically relevant micromolar to submicromolar range. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific variants in a preferred orientation. 3D atomic models reveal that such preference in orientation results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues within the basic regions of Jun and Fos. I further demonstrate that binding of the biologically relevant Jun-Jun homodimer to TRE and CRE occurs with favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures in a manner akin to the binding of Jun-Fos heterodimer. However, anomalously large negative heat capacity changes provoke a model whereby Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. The data also reveal that the heterodimerization of leucine zippers is modulated by the basic regions and these regions may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain a-helical conformation in isolation and the formation of the native coiled coil a-helical dimer is attained through a coupled folding-dimerization mechanism. Taken together, this dissertation marks the first comprehensive thermodynamic analysis of an otherwise well-studied and vitally important transcription factor. My studies shed new light on the forces driving the AP1-DNA interaction in thermodynamic and structural terms. The implications of these novel findings on the development of novel therapies for the treatment of disease with greater efficacy coupled with low toxicity cannot be overemphasized.
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Fellinger, Karin. "Analysis of Protein Interactions Controlling DNA Methyltransferases." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-98919.
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Russom, Aman. "Microfluidic bead-based methods for DNA analysis." Doctoral thesis, KTH, Skolan för elektro- och systemteknik (EES), 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-155.
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With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods. This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed. The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers. The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides.QC 20101008
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Edlund, Hanna. "Sensitive Identification Tools in Forensic DNA Analysis." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-131904.
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DNA as forensic evidence is valuable in criminal investigations. Implementation of new, sensitive and fast technologies is an important part of forensic genetic research. This thesis aims to evaluate new sensitive methods to apply in forensic DNA analysis including analysis of old skeletal remains. In Paper I and II, two novel systems for analysis of STRs, based on the Pyrosequencing technology, are presented. In Paper I, Y chromosomal STRs are analysed. Markers on the male specific Y chromosome are especially useful in analysis of DNA mixtures. In Paper II, ten autosomal STRs are genotyped. The systems are based on sequencing of STR loci instead of size determination of STR fragments as in routine analysis. This provides a higher resolution since sequence variants within the repeats can be detected. Determination of alleles is based on a termination recognition base. This is the base in the template strand that is excluded from the dispensation order in the sequencing of the complementary strand and therefore terminates the reaction. Furthermore, skeletal remains are often difficult to analyse, due to damaging effects from the surrounding environment on the DNA and the high risk of exogenous contamination. Analysis of mitochondrial DNA is useful on degraded samples and in Paper III, mtDNA analysis of 700 years old skeletal remains is performed to investigate a maternal relationship. The quantity and quality of DNA are essential in forensic genetics. In Paper IV the efficiency of DNA isolation is investigated. Soaking skeletal remains in bleach is efficient for decontamination but result in a lower DNA yield, especially on pulverised skull samples. In conclusion, this thesis presents novel sequencing systems for accurate and fast analysis of STR loci that can be useful in evaluation of new loci and database assembly as well as the utility of mtDNA in forensic genetics.
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Freeman, Emmerson Clare. "Molecular analysis of mutant human mitochondrial DNA." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297942.
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Hon, Wing-hong, and 韓永康. "Analysis of DNA shuffling by computer simulation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B27771027.
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高銘謙 and Ming-him Ko. "A multi-agent model for DNA analysis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B31222778.
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Shahran, M. K. "The analysis of flow cytometric DNA data." Thesis, University of Nottingham, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371132.
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Routledge, Michael Norman. "'3'2p-Postlabelling analysis of carcinogen DNA adducts." Thesis, University of York, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292540.
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Ghorbani, Mohammadmersad. "Computational analysis of CpG site DNA methylation." Thesis, Brunel University, 2013. http://bura.brunel.ac.uk/handle/2438/8217.
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Epigenetics is the study of factors that can change DNA and passed to next generation without change to DNA sequence. DNA methylation is one of the categories of epigenetic change. DNA methylation is the attachment of methyl group (CH3) to DNA. Most of the time it occurs in the sequences that G is followed by C known as CpG sites and by addition of methyl to the cytosine residue. As science and technology progress new data are available about individual’s DNA methylation profile in different conditions. Also new features discovered that can have role in DNA methylation. The availability of new data on DNA methylation and other features of DNA provide challenge to bioinformatics and the opportunity to discover new knowledge from existing data. In this research multiple data series were used to identify classes of methylation DNA to CpG sites. These classes are a) Never methylated CpG sites,b) Always methylated CpG sites, c) Methylated CpG sites in cancer/disease samples and non-methylated in normal samples d) Methylated CpG sites in normal samples and non-methylated in cancer/disease samples. After identification of these sites and their classes, an analysis was carried out to find the features which can better classify these sites a matrix of features was generated using four applications in EMBOSS software suite. Features matrix was also generated using the gUse/WS-PGRADE portal workflow system. In order to do this each of the four applications were grid enabled and ported to BOINC platform. The gUse portal was connected to the BOINC project via 3G-bridge. Each node in the workflow created portion of matrix and then these portions were combined together to create final matrix. This final feature matrix used in a hill climbing workflow. Hill climbing node was a JAVA program ported to BOINC platform. A Hill climbing search workflow was used to search for a subset of features that are better at classifying the CpG sites using 5 different measurements and three different classification methods: support vector machine, naïve bayes and J48 decision tree. Using this approach the hill climbing search found the models which contain less than half the number of features and better classification results. It is also been demonstrated that using gUse/WS-PGRADE workflow system can provide a modular way of feature generation so adding new feature generator application can be done without changing other parts. It is also shown that using grid enabled applications can speedup both feature generation and feature subset selection. The approach used in this research for distributed workflow based feature generation is not restricted to this study and can be applied in other studies that involve feature generation. The approach also needs multiple binaries to generate portions of features. The grid enabled hill climbing search application can also be used in different context as it only requires to follow the same format of feature matrix.
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Odell, Mark. "An analysis of vaccinia virus DNA ligase." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670258.
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Van, Winkle Carolyn. "Forensic DNA Extraction Strategies for PCR Analysis." Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc278269/.
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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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Yoshida, Kanako. "Analysis of DNA polymorphisms for human identification." Kyoto University, 2000. http://hdl.handle.net/2433/181418.
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Ko, Ming-him. "A multi-agent model for DNA analysis /." Hong Kong : University of Hong Kong, 1999. http://sunzi.lib.hku.hk/hkuto/record.jsp?B21949116.
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Joseph, Ansamma K. "DNA sequence analysis of T cell receptors." Thesis, University of Bath, 1996. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321849.
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Baccouche, Alexandre. "Functional analysis of artificial DNA reaction network." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB135/document.
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La gestion et transmission d’information au sein d’organismes vivants implique la production et le trafic de molécules via des voies de signalisation sutructurées en réseaux de réactions chimiques. Ces derniers varient selon leur forme, taille ainsi que la nature des molécules mises en jeu. Parmi eux, les réseaux de régulation génétiques nous ont servi de modèle pour le développement et la mise en place d’un système de programmation moléculaire in vitro. En effet, l’expression d’un gène est majoritairement dominé par des facteurs de transcription, autres protéines ou acides nucléiques, eux-mêmes exprimés par d’autres gènes. L’ensemble forme l’interactome de la cellule, carte globale des interactions entre gènes et sous-produits, où la fonction du réseau est relié à sa topologie. L’observation des noeuds et sous-architectures dénote trois mécanismes récurrents : premièrement, la nature des interactions est de type activation ou inhibition, ce qui implique que tout comportement non trivial est obtenu par une combinaison de noeuds plutôt que le développement de nouvelles interactions. Ensuite, la longévité du réseau est assurée par la stabilité chimique de l’ADN couplée à la chimiosélectivié des réactions enzymatiques. Enfin, l’aspect dynamique est maintenu par le constant anabolisme/catabolisme des intermédiaires et donc l’utilisation de combustible/énergie. C’est suivant ces observations que nous avons développé un ensemble de trois réactions enzymatiques élémentaires : la «PEN-DNA toolbox». L’architecture du réseau, à savoir les connections entre les noeuds est médiée par la séquence de brins d’ADN synthétiques (appelés matrice), et trois enzymes (polymérase, nickase, et exonucléase) assurent la catalyse des réactions chimiques. La production et dégradation des intermédiaires consomme des désoxyribonucléotides triphosphates et rejette des désoxyribonucléotides monophosphate, dissipant ainsi le potentiel chimique. Les réactions sont suivies grâce au greffage d’un fluorophore sur le brin matriciel et au «nucleobase quenching» qui intervient lorsqu’une base d’un intermédiaire se rapproche du fluorophore après hybdridation sur le brin matriciel. L’activation correspond alors à la synthèse d’un brin output en réponse à un brin input, alors que l’inhibition survient lorsqu’un brin output s’hybride sur un brin matriciel, empêchant ainsi à l’input correspondant de s’y fixer. Oscillations, bistabilité et mémoire sont des exemples de comportements implémentés en PEN-DNA toolbox, faisant appel à des architectures de plus en plus complexes. Pour cela, un réglage fin des concentrations en effecteurs (ADN et enzymes) est nécessaire, ce qui sous-tend l’existence de plusieurs comportements pour un même circuit, dépendant des conditions paramétriques. L’établissement d’une carte de chaque combinaison de paramètres avec le comportement global associé permettrait de comprendre le fonctionnement du réseau dans son ensemble, et donnerait accès à tous les comportements disponibles. Dans le cas d’un système dynamique non linéaire, une telle carte est un diagramme de bifurcation du système. Pour explorer de manière exhaustive les possibilités d’un réseau dans un cadre expérimental raisonable, nous avons développé une plateforme microfluidique capable de générer des goutelettes d’eau dans l’huile à partir de quatres canaux aqueux différents. Ce dispositif nous donne accès, grâce à un contrôle fin des contributions de chaque canal aqueux, à des goutelettes monodisperses (volume de l’ordre du picolitre) dont le contenu est différent pour chaque goutelette. Nous avons adapté notre dispositif aux contraintes matérielles (design microfluidique, génération de goutelettes à contenu différents et controllés, observation et stabilité à long terme) et techniques (tracabilité des goutelettes et stabilité/compatibilité chimique). (...)Information processing within and in between living organisms involves the production and exchange of molecules through signaling pathways organized in chemical reactions networks. They are various by their shape, size, and by the nature of the molecules embroiled. Among them, gene regulatory networks were our inspiration to develop and implement a new framework for in-vitro molecular programming. Indeed, the expression of a gene is mostly controlled by transcription factors or regulatory proteins and/or nucleic acids that are themselves triggered by other genes. The whole assembly draws a web of cross-interacting genes and their subproducts, in which the well controlled topology relates to a precise function. With a closer look at the links between nodes in such architectures, we identify three key points in the inner operating system. First, the interactions either activate or inhibit the production of the later node, meaning that non trivial behaviors are obtained by a combination of nodes rather than a specific new interaction. Second, the chemical stability of DNA, together with the precise reactivity of enzymes ensures the longevity of the network. Finally, the dynamics are sustained by the constant anabolism/catabolism of the effectors, and the subsequent use of fuel/energy. All together, these observations led us to develop an original set of 3 elementary enzymatic reactions: the PEN-DNA toolbox. The architecture of the assembly, i.e. the connectivity between nodes relies on the sequence of synthetic DNA strands (called DNA templates), and 3 enzymes (a polymerase, a nickase and an exonuclease) are taking care of catalysis. The production and degradation of intermediates consume deoxyribonucleoside triphosphates (dNTP) and produce deoxynucleotide monophosphates leading to the dissipation of chemical potential. Reactions are monitored thanks to a backbone modification of a template with a fluorophore and the nucleobase quenching effect consecutive to an input strand binding the template. The activation mechanism is then the production of an output following the triggering of an input strand, and the inhibition comes from the production of an output strand that binds the activator-producing sequence. Various behaviors such as oscillation, bistability, or switchable memory have been implemented, requiring more and more complex topologies. For that, each circuit requires a fine tuning in the amount of chemical parameters, such as templates and enzymes. This underlies the fact that a given network may lead to different demeanors depending on the set of parameters. Mapping the output of each combination in the parameter space to find out the panel of behaviors leads to the bifurcation diagram of the system. In order to explore exhaustively the possibilities of one circuit with a reasonable experimental cost, we developped a microfluidic tool generating picoliter-sized water-in-oil droplets with different contents. We overcame the technical challenges in hardware (microfluidic design, droplet generation and long-term observation) and wetware (tracability of the droplet and emulsion compatibility/stability). So far, bifurcation diagrams were calculated from mathematical models based on the enzymes kinetics and the thermodynamic properties of each reaction. The model was then fitted with experimental data taken in distant points in the parameter space. Here, millions of droplets are created, and each one encloses a given amount of parameters, becoming one point in the diagram. The parameter coordinates are barcoded in the droplet, and the output fluorescence signal is recorded by time lapse microscopy. We first applied this technique to a well-known network, and obtained the first experimental two-dimensional bifurcation diagram of the bistable system. The diagram enlightens features that were not described by the previous mathematical model. (...)
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LAPI, MICHELA. "STRUCTURAL ANALYSIS OF TRANSCRIPTION FACTOR/DNA COMPLEXES." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/834212.
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Binding of transcription factors (TFs) to discrete sequences in gene promoters and enhancers is crucial to the process by which genetic information is transferred to biological functions. TF structural analysis is the key to understanding their DNA-binding mode and for the design of specific inhibitors. In this context, the present PhD project focuses on two TFs: (1) NFIX, a TF with unknown structure that binds to the palindromic motif TTGGC(n5)GCCAA and plays an essential role in skeletal muscle development; and (2) NF-Y, a histone-like TF that binds the CCAAT box in promoters of cell cycle genes.
(1) There is a lack of structural information on NFIX and relative TF family members because of the challenge expression and purification of soluble protein constructs in the amount required for structural characterization. We were able to obtain functional NFIX constructs using E. coli cells, and to purify them with a high yield. NFIX constructs were tested for correct folding, stability, and DNA-binding through a series of biochemical and biophysical methods. Furthermore, we managed to produce well-diffracting NFIX crystals, which were used for Single Anomalous Diffraction (SAD) phasing. We collected two different datasets of same NFIX construct at 2.7 Å and 3.5 Å resolution, in two different space groups. Structural analysis of this NFIX construct shed first light on this class of TFs and put the bases for the understanding of its DNA-binding mode.
(2) Genomic data of NF-Y locations at gene promoters indicate that NF-Y plays a key role in oncogenic activation. The knowledge of the 3D structure of NF-Y in complex with its CCAAT box provided the rationale for developing inhibitors able to interfere with DNA-binding. The pipeline used to search for NF-Y inhibitors consisted of in silico screenings of compounds that interfere with NF-Y functional trimerization and/or with CCAAT box interaction, followed by in vitro biochemical/biophysical confirmation of inhibition and X-ray crystallography validation. The selected compound from the initial screening was suramin, which proved to bind to NF-Y and to functionally inhibit the binding of DNA. We obtained suramin/protein complex co-crystals, which diffracted up to 2.3 Å resolution. The crystal structure of the suramin/NF-Y provides the first evidence of NF-Y inhibition by a small molecule.