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Showing 31 to 40 of 43 (0.244 seconds)Spelling suggestions: "subject:"allelespecific"" "subject:"allergenspecific""
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Optimization of a multiplex ARMS-PCR for detection of the primary mutations causing Leber’s hereditary optic neuropathJäder, Klara January 2020 (has links)
Leber’s hereditary optic neuropathy (LHON) is a genetic disease that causes the patients to become blind, first in one eye and then the other, around the ages of 10-75 years. The disease is caused by mutations in the mitochondrial DNA, which disturbs the respiratory chain leading to the deterioration of the retinal ganglion cells. This study’s aim is to optimize a multiplex amplification-refractory mutation system PCR for detection of three primary mutations causing LHON. This was done through a series of PCRs, including PCR aimed at the ß-globin gene, conventional simplex PCR and a simplex ARMS-PCR aimed at the three primary mutations causing LHON. This study was, however, terminated prematurely due the Covid-19 outbreak and the optimization of the ARMS-PCR could therefore not be done. This study’s aim was adapted to the new circumstances to instead provide guidance on how to perform the optimization using the results from the PCRs that were done before the termination. The results found that for the ARMS-PCR 2 mM of magnesium would suffice as a start point overall and the need to solve the problems with the two 14484 plasmids was evident. The ARMS-PCR is one of many methods that can be used to the detect single nucleotide polymorphism, but its availability and robustness makes this a method worth optimizing. To continue with the optimization of the ARMS-PCR several factors would have to be tested, including annealing temperature, primer concentrations and magnesium concentration.
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The Identification of Colorectal Cancer Susceptibility Genes Using a Cross-Species, Systems Genetics ApproachGerber, Madelyn Margaret 19 May 2015 (has links)
No description available.
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Cis-regulatory variation and divergence in CapsellaSteige, Kim A. January 2016 (has links)
Cis-regulatory changes in e.g. promoters or enhancers that affect the expression of a linked focal gene have long been thought to be important for adaptation. In this thesis, I investigate the selective importance and genomic correlates of cis-regulatory variation and divergence in the genus Capsella, using massively parallel sequencing data. This genus provides an opportunity to investigate cis-regulatory changes in response to polyploidization and mating system shifts, as it harbors three diploid species, the outcrosser Capsella grandiflora and the selfers Capsella orientalis and Capsella rubella, as well as the tetraploid Capsella bursa-pastoris. We first identify cis-regulatory changes associated with adaptive floral evolution in connection with the recent switch to self-fertilization in C. rubella and show that cis-regulatory changes between C. rubella and its outcrossing close relative C. grandiflora are associated with differences in transposable element content. Second, we show that variation in positive and purifying selection is important for the distribution of cis-regulatory variation across the genome of C. grandiflora. Interestingly, the presence of polymorphic transposable elements is strongly associated with cis-regulatory variation in C. grandiflora. Third, we show that the tetraploid C. bursa-pastoris is of hybrid origin and investigate the contribution of both parental species to gene expression. We show that gene expression in the tetraploid is partly explained by cis-regulatory divergence between the parental species. Nonetheless, within C. bursa-pastoris there is a great deal of variation in homeolog expression. In summary, this thesis explores the role of cis-regulatory changes for adaptive morphological changes in connection to a shift in mating system, the role of cis-regulatory divergence between progenitor species for an allopolyploid as well as the impact of positive and purifying selection on cis-regulatory variation within a species.
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Establishment of a Y-chromosome specific extraction method for the separation of Y-chromosomal haplotypes from male DNA mixturesRothe, Jessica 05 June 2014 (has links)
Die Haplotypspezifische Extraktion (HSE) bietet für die Analyse von männlichen Mischprofilen einen neuen und direkteren Lösungsansatz, in dem die haploiden Y-chromosomalen DNS-Komponenten der einzelnen Individuen bereits vor der Analyse der individual spezifischen Marker separiert werden können und dadurch eine wirkliche physische Trennung erreicht wird. Die HSE verwendet Y-chromosomalen SNPs für die Erstellung allelspezifische Extraktionssonden, die nun gezielt nur die Marker der extrahierten DNS Komponente bzw. einer Person separieren sollen. Dabei werden im Hybridisationsschritt der HSE selektive nur komplett hybridisierte Sonden durch eine Polymerase verlängert. Während der Elongation erfolgt eine Biotinylierung des neu entstehenden Stranges, welcher dann selektiv durch Streptavidin markierte Eisenkügelchen extrahiert werden kann. Erste Durchführungen einer HSE zeigten nur eine sehr schwache bis keine Anreicherung. Während der Optimierung verschiedener Parameter wurde die Schlüsselstellung des Sondendesigns in der HSE-Technik deutlich. Die Ergebnisse zeigten, dass die neu entwickelten Sonden den Trennungserfolg der Mischprobe enorm verbessern und in einigen Fällen sogar zum Ausschluss des konkurrierenden Allels führten. Ein Vergleich der HSE Ergebnisse mit den simulierten Sondenparametern der getesteten Sonden ergab, dass der Extraktionserfolg der Sonde maßgeblich durch das Zusammenspiel von Sondenlänge und GC-Gehalt bestimmt wird. Durch dieses neu gewonnene Verständnis über den Einfluss der einzelnen Sondenparameter auf den Trennungserfolg der Mischprobe, können für künftige HSE Anwendungen Sonden effizienter erstellt und deren Wirksamkeit vorhergesagt werden. Zusätzlich konnte das neu entwickelte Vorhersage-Model der Sondenspezifität auch für weitere Extraktionsorte außerhalb des Y-Chromosoms bestätigt werden. Weiterhin konnte durch die Kombination verschiedener Sonden in einer Multiplex HSE mehrerer Y-chromosomaler Marker gleichzeitig getrennt. / Haplotype-specific extraction (HSE) allows the separation of diploid samples in their haploid components and offers in forensic a new straight forward method to separate Y-chromosomal mixed profiles, consisting of haplotype markers like short tandem repeats (STRs). The advantage of the HSE approach in mixture analysis is the real physical separation of the individual DNA components before the amplification of the STR markers. In order to use the HSE technique for the separation of male DNA mixtures, Y-chromosomal extraction probes were designed to single nucleotide polymorphisms (SNPs), which have been specific for one contributor of the male DNA mixtures. During extraction only complete matched probes are extended by a polymerase which results in the incorporation of biotinylated nucleotides. The synthesized and biotin labeled strand is separated by streptavidin coated magnetic beads. Finally, samples were analyzed by PCR coupled capillary electrophoresis for the detection of the extracted STR markers. First tests of a Y-chromosomal specific extraction showed only little till no enrichment of the targeted alleles. Therefore optimization tests of different parameters were carried out, which revealed the probe design as the key factor of successful HSE. A comparison between simulated probe parameters and their extraction success in HSE showed that the HSE probe efficiency mainly depends of the relation of probe length and GC-contents. Because of the new gained knowledge about the influence of the probe-design on the separation success, probes for future HSE application can be developed faster and cost-effective. The new prediction model for probe-specificity was also successful tested for the extraction of other genome-loci. Furthermore, a multiplex HSE approach was used to separate several STR markers simultaneously in one extraction reaction and therefore achieved the separation of one contributor Y-chromosomal haplotype.
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Microfluidic bead-based methods for DNA analysisRussom, Aman January 2005 (has links)
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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A Novel Approach to Identify Candidate Imprinted Genes in HumansShapiro, Jonathan 21 March 2012 (has links)
Many imprinted genes are necessary for normal human development. Approximately 70 imprinted genes have been identified in humans. I developed a novel approach to identify candidate imprinted genes in humans using the premise that imprinted genes are often associated with nearby parent-of-origin-specific DNA differentially methylated regions (DMRs). I identified parent-of-origin-specific DMRs using sodium bisulfite-based DNA (CpG) methylation profiling of uniparental tissues, mature cystic ovarian teratoma (MCT) and androgenetic complete hydatidiform mole (AnCHM), and biparental tissues, blood and placenta. In support of this approach, the CpG methylation profiling led to the identification of parent-of-origin-specific differentially methylated CpG sites (DMCpGs) in known parent-of-origin-specific DMRs. I found new DMRs for known imprinted genes NAP1L5 and ZNF597. Most importantly, I discovered many new DMCpGs, which were associated with nearby genes, i.e., candidate imprinted genes. Allelic expression analyses of one candidate imprinted gene, AXL, suggested polymorphic imprinting of AXL in human blood.
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A Novel Approach to Identify Candidate Imprinted Genes in HumansShapiro, Jonathan 21 March 2012 (has links)
Many imprinted genes are necessary for normal human development. Approximately 70 imprinted genes have been identified in humans. I developed a novel approach to identify candidate imprinted genes in humans using the premise that imprinted genes are often associated with nearby parent-of-origin-specific DNA differentially methylated regions (DMRs). I identified parent-of-origin-specific DMRs using sodium bisulfite-based DNA (CpG) methylation profiling of uniparental tissues, mature cystic ovarian teratoma (MCT) and androgenetic complete hydatidiform mole (AnCHM), and biparental tissues, blood and placenta. In support of this approach, the CpG methylation profiling led to the identification of parent-of-origin-specific differentially methylated CpG sites (DMCpGs) in known parent-of-origin-specific DMRs. I found new DMRs for known imprinted genes NAP1L5 and ZNF597. Most importantly, I discovered many new DMCpGs, which were associated with nearby genes, i.e., candidate imprinted genes. Allelic expression analyses of one candidate imprinted gene, AXL, suggested polymorphic imprinting of AXL in human blood.
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Molecular Marker Applications in Oat (Avena Sativa L.) Breeding and Germplasm DiagnosticsBenazir Katarina, Marquez 27 May 2014 (has links)
The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.
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Molecular Marker Applications in Oat (Avena Sativa L.) Breeding and Germplasm DiagnosticsBenazir Katarina, Marquez January 2014 (has links)
The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.
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Chromatin folding in health and disease: exploring allele-specific topologies and the reorganization due to the 16p11.2 deletion in autism-spectrum disorder.Kempfer, Rieke 09 November 2020 (has links)
Die 3D Struktur von Chromosomen im Zellkern reguliert verschiedene Funktionen in der Zelle und Fehler in der 3D Faltung des Genoms können pathogen sein. 3D Genomfaltung kann mit verschiedenen Methoden untersucht werden um Chromatinkontakte, sowie die Position von DNA in Relation zu sub-nuklearen Bereichen oder der Kernmembran zu detektieren. Hier verwende ich GAM und Hi-C um zwei Aspekte der 3D Genomtopologie zu untersuchen, die Allelspezifität von Chromatinkontakten und Kontakte zwischen Chromosomen. Ich untersuche allelspezifische Kontakte in murinen embryonalen Stammzellen und Interaktionen zwischen Chromosomen im Zusammenhang mit Autismus Spektrum Störung auf ihre Relevanz in der Regulation von Genen.
Zur allelspezifischen Detektion von Chromatinkontakten generierte ich einen GAM Datensatz der tausende von nuklearen Cryodünnschnitten enthält. Die Generierung dieser Daten beinhaltete die Entwicklung einer verbesserten Version der GAM Methode zur Produktion von großen Datensätzen in Hochdurchsatz. Hier zeige ich, dass GAM effizient Haplotyp-spezifische Chromatinkontakte bestimmen kann. Erste Untersuchungen von allelspezifischer 3D Genomtopologie zeigten weitreichende Unterschiede zwischen den Allelen, welche „A/B compartments“ und spezifische Chromatinkontakte beinhalten, wie zum Beispiel am Imprinting Locus H19/Igf2.
Zur Untersuchung von interchromosomalen Kontakten detektierte ich Chromatinkontakte mit Hi-C im Kontext einer genomischen Deletion am humanen 16p11.2 Locus, assoziiert mit Autismus Spektrum Störung. Ich zeige hier, dass die Deletion am 16p11.2 Locus zu der Reorganisation von spezifischen interchromosomalen Kontakten zwischen 16p11.2 und Chromosom 18 führt, und stelle eine Hypothese auf wie diese interchromosomalen Kontakte zur ektopischen Aktivierung von Pcdh Genen auf Chromosom 18 führen. Protocadherins haben wichtige Funktionen in neuronaler Konnektivität, ein Prozess dessen Störung zur Manifestierung von Autismus Spektrum Störung beitragen könnte. / The 3D folding of interphase chromosomes inside the nucleus regulates important nuclear functions and once disrupted can lead to the manifestation of disease. Different techniques can be used to map 3D genome folding and detect pairwise and multiway interactions of the genome, or map the positions of DNA with respect to subnuclear compartments or the nuclear lamina. Here, I use GAM and Hi-C to explore two aspects of 3D genome topology, the allele specificity of chromatin contacts and long-range contacts between chromosomes, respectively. I detect specific contacts of the parental alleles in mouse embryonic stem cells and interactions between chromosomes in the context of congenital disease and study them with regard to their functionality and importance in mammalian gene regulation.
For detecting chromatin contacts with allele specificity, I produced a GAM dataset containing thousands of nuclear slices. The collection of this data was accompanied by the development of a high-throughput version of GAM that allows the generation of large datasets. I show that GAM can determine haplotype-specific chromatin contacts with high efficiencies. First explorations of allele-specific chromatin topologies reveal many differences between the parental alleles, including allele-specific compartments A and B, and specific chromatin contacts, for example at the imprinted H19/Igf2 locus.
For the exploration of inter-chromosomal contacts in disease, I mapped chromatin interactions with Hi-C in the context of a CNV at the human 16p11.2 locus, associated with autism spectrum disorders. Here, I show that the deletion at the 16p11.2 locus results in the rearrangement of specific inter-chromosomal contacts between the 16p11.2 locus and chromosome 18 and propose a role for these inter-chromosomal contact changes in the upregulation of the nearby Pcdhb gene cluster, which comprises protocadherin genes with important functions in neuronal connectivity during development.
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