Browsing by Subject "qPCR"

Now showing 1 - 6 of 6
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    DNA-based detection and reference genome assembly of Aphanomyces cochlioides
    (2021-08) Botkin , Jacob
    Aphanomyces root rot (ARR) and Aphanomyces damping-off, caused by the soil-borne oomycete A. cochlioides, are common diseases of sugar beet in major production regions. Management techniques are implemented to mitigate losses, but ultimately A. cochlioides is intractable and significantly reduces the sucrose content of the sugar beet taproot throughout the growing season, especially during periods with above average precipitation. Currently, a genome sequence for A. cochlioides is not available, and existing diagnostic assays are time consuming and have limitations. The first objective was to assemble and annotate a reference genome for A. cochlioides. We conducted a de novo genome assembly and annotation of A. cochlioides using 232x coverage of Nanopore long-reads, and error corrected with 77x coverage of Illumina short-reads. The assembled genome was 76.3 Mb, consisted of 97 contigs, and had a contig N50 of 2.6 Mb. The assembly contained 93.2% of complete benchmarking universal single-copy orthologs, a repeat content of 32.1%, and 20,274 gene models. This is the first report of a reference genome for A. cochlioides, which could serve as a platform for future investigations into virulence mechanisms, comparative genomics, and the development of diagnostic assays. The second objective was to develop a rapid, sensitive, and accurate DNA-based detection assay to quantify A. cochlioides inoculum in infested soil and infected sugar beet tissue. We developed a TaqMan qPCR assay that was specific to A. cochlioides. The qPCR assay was validated with 12 naturally infested soil samples, which had Ct (cycle threshold) values of 26.72 to 34.64 and ARR disease severity index (DSI) values of 48 to 100. The qPCR assay was further validated on infected adult sugar beets and seedlings. For 60 adult sugar beet roots, A. cochlioides DNA was detected in 63% of the samples, while a culture-based assay identified A. cochlioides in 15% of the samples. Furthermore, A. cochlioides DNA was detected in infected seedlings as early as 5 days after planting in a naturally infested soil. Finally, when oospore infested potting soil was tested with the qPCR assay and ARR bioassay, a strong correlation was observed between oospore density and Ct value (R2 = 0.96), as well as oospore density and DSI value (R2 = 0.968). The limit of detection (LOD) was 5 oospores per g soil (dry wt.), which had a mean Ct value of 34.58, and a mean DSI value of 23.33. Our DNA-based detection assay could provide growers with the A. cochlioides infestation level of field soils to help them make informed management decisions prior to planting.
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    Establishing MapR: A Genome-wide R-loop Mapping Strategy
    (2021-06) Zhang, Zijun
    R-loops are transcription intermediates containing an RNA:DNA hybrid and a displaced single-strand DNA. R-loops are important regulators of various cellular processes such as transcription and DNA repair. However, when R-loop levels and/or distributions in the genome are dysregulated, they act as endogenous sources of genomic instability, a hallmark of cancers. Mutations in genes encoding for R-loop regulators are observed in various cancers and diseases, linking R-loop dysregulation to disease pathogenesis. As the number of known R-loop regulators increases, this raises an intriguing question on how different factors contribute to R-loop homeostasis. Furthermore, whether various R-loop regulators function at the same R-loop regions in different diseases is largely unknown. In order to answer these questions, a proper mapping method to analyze R-loop landscapes in different diseases will be necessary. My thesis project focuses on establishing an R-loop mapping strategy called MapR. MapR was reported to produce a high signal-to-noise ratio in genome-wide next-generation sequencing (NGS). However, it lacks a quality control step prior to NGS. In this study, I successfully established and optimized the MapR method to quantify R-loop levels by quantitative PCR (MapR-qPCR) as the quality control step. A significantly higher level of R-loop enrichment was observed at the R-loop positive RPL13A locus compared to that at the R-loop negative SNRPN locus by the MapR-qPCR analysis. Furthermore, treating MapR samples with RNaseH, an enzyme that hydrolyzes the RNA moiety within RNA:DNA hybrids, partially suppressed R-loop enrichment at RPL13A locus, suggests that MapR enriches for R-loops. My project will allow us to integrate the MapR-qPCR analysis as quality control checkpoints prior to genome-wide sequencing.
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    Light-mediated Sexual Dimorphism in Opsin Expression During Spawning in Nematostella vectensis
    (2024-04) Wagner, Starla J.; McCulloch, Kyle J.
    Across animals, opsins are the primary protein responsible for light detection. Currently, there is a large gap in knowledge in the evolutionary history of opsin function and how it correlates with other biological responses like spawning. Cnidarians (jellyfish and anemones) are prime candidates for closing this gap. They are a sister taxon to bilaterally symmetric animals like flies and humans, and so studying their opsin function and expression in non-visual contexts allows for further understanding of how light sensing may have evolved to form modern visual systems. In this experiment, qPCR analysis on the Cnidarian, Nematostella vectensis (the starlet sea anemone), was used to determine the effect of certain wavelengths of light that an animal was exposed to during spawning had on opsin expression levels. The impact of sex and tissue type on these expression levels was an additional area of interest. The data showed that certain wavelengths like blue light were correlated with larger amounts of opsin expression in female mesenteries and tentacles/skin tissue than in male tissue types. This indicates that opsin expression is sexually dimorphic which implies there is a relationship between opsin expression and spawning, something that was previously unknown. Future experiments using RNA-seq will allow for a deeper understanding of this relationship and the proteins involved.
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    Pathogen Quantification and Risk Assessment of Water Reuse Systems in the State of Minnesota
    (2019-12) Dooling, Valerie
    There is a growing interest in reusing or reclaiming water for non-potable use; however, one of the largest barriers to implementation is unknown source water quality and what risks are present. This work was conducted to determine the number of pathogens and their concentration present in currently operational water reuse systems in Minnesota, and to collect design treatment information from each reuse site and compare to pathogen and indicator quantity. Eighty-three samples were collected from 25 sites and were simultaneously analyzed for 27 bacterial and viral genes through microfluidic qPCR. Findings are that indicators and chemical tests did not correlate with type of source water. Type of treatment does not consistently correlate to log10 reduction, and only wastewater disinfection consistently removed all pathogens from treatment. Ten different pathogens were detected in water reuse systems. Quantitative Microbial Risk Assessment was performed on six pathogens in four exposure scenarios. Findings are that annual risk of infection of non-treated water is greater than recreational water benchmarks, however, the extent of risks depend on the exposure. When only considering samples from distribution or post-treatment sites, the risks are considerably lower, and often within guidelines.
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    Repair of DNA-protein crosslinks in mammalian cells
    (2018-07) Chesner, Lisa
    The work below describes a new assay called strand-specific primer extension-quantitative polymerase chain reaction (SSPE-qPCR) used to study the repair of DNA-protein crosslinks in mammalian cells. DNA-protein crosslinks (DPCs) are bulky lesions which disrupt important cell processes such as transcription and replication. They are formed by endogenous molecules such as formaldehyde and exogenous damaging agents such as ionizing radiation. However, the repair mechanisms associated with their repair are still unclear. Chapter 1 of this document provides background information on the formation, biological consequences, current models, and methods used to study DPC repair. Chapter 2 describes the SSPE-qPCR assay and its uses/limitations for studying the repair of plasmids containing DPCs or other polymerase-blocking adducts transfected into mammalian cells. Chapter 3 describes results generated using this assay to assess the role of nucleotide excision repair in DPC repair and highlights the versatility of the SSPE-qPCR assay. Chapter 4 extends observations made in Chapter 3 by using SSPE-qPCR to examine repair of DPC-containing plasmids in the presence of a homologous donor. It also provides evidence for homologous recombinational repair of DPCs in mammalian mitochondria. Overall, this work provides additional insight into the mechanisms of DPC repair in the nucleus and mitochondria using a quantitative, flexible assay that has not been available previously.
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    Temporal and Spatial Trends in the Abundance of Functional Denitrification Genes and Observed Soil Moisture and Potential Denitrification Rates.
    (2016-09) Lurndahl, Nicole
    In an attempt to identify a genetic basis for observed denitrification rates, denitrification functional genes nirS, nirK, narG, cnorBB, nosZ1, and nosZ3 were quantified using qPCR of DNA extracted from soils and sediments taken from agricultural runoff ditches in the Seven Mile Creek watershed. Gene copy numbers were analyzed with respect to both spatial and temporal scales, as well as compared to soil moisture levels and potential denitrification as determined by the acetylene inhibition method. Results of this study suggest a link between soil moisture and potential denitrification, although more formal relationships could be determined. Discriminant analyses also show similarities in genetic composition of analyzed samples over both spatial and temporal scales.