Theses and Dissertations at Montana State University (MSU)
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Item Development of drop-based microfluidic methods for high-throughput biological assays(Montana State University - Bozeman, College of Engineering, 2021) Zath, Geoffrey Kane; Chairperson, Graduate Committee: Connie Chang; This is a manuscript style paper that includes co-authored chapters.Drop-based microfluidics allows single-cell biological assays to be performed by encapsulating samples in picoliter scale drops. Adapting biological assays to drop-based microfluidics requires novel approaches to meet the method requirements of each assay. For example, microtiter plates are a common tool for storing many unique samples in some assays. An equivalent strategy for drops involves labeling samples with a barcode prior to drop encapsulation and storing the barcoded drops in a single mixture, thereby creating a drop library. Other assay adaptions, such as drop-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) require that drops be stabilized during the high temperatures used for thermal cycling. Drop-based RT-qPCR is useful for studying single-cell dynamics in drops, such as influenza A virus (IAV) infection. Conventional methods for measuring IAV output from individual cells are labor intensive and low-throughput. Thus, there is a need to adapt RT-qPCR to drop-based microfluidics for the purpose of high-throughput single cell analysis of infected cells. The research presented here focuses on the characterization of the Pressure Cooker Chip (PCC) to rapidly encapsulate drop libraries and the development of a drop-based RT-qPCR method to measure IAV output from infected cells. The PCC was used to make drop libraries by rapidly generating drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard microtiter well plate. The drop library was optically barcoded using a two-color combination of fluorescent microbeads or quantum dots with 24 or 192 unique combinations, respectively. To adapt RT-qPCR in drops, known PCR additives were systematically tested to optimize drop stability and limit dye diffusion during thermocycling. A novel qPCR data analysis method was developed to convert drop fluorescence data collected at a single thermocycle to an initial RNA template concentration. Together, the additive screening and novel qPCR data analsyis method enabled the use of drop-based RT-qPCR to quantify the highly heterogeneous IAV burst size from single cells in thousands of drops. Our method is the first to measure single cell IAV burst size using a high-throughput, drop-based RT-qPCR assay.Item A polymerase chain reaction for the detection of the take-all fungus, Gaeumannomyces graminis, in infected wheat seedlings(Montana State University - Bozeman, College of Agriculture, 1990) Schesser, Kurt RobertItem Detection of Ustilago hordei in barley leaf tissue by polymerase chain reaction and analysis of the MAT-2 pheromone and pheromone receptor genes(Montana State University - Bozeman, College of Agriculture, 1998) Willits, Deborah AnnItem Application of PCR to detect varieted purity in barley malt(Montana State University - Bozeman, College of Agriculture, 1997) Habernicht, Debra KayItem Selection and characterization of genomic DNA clones of Pyrenophora teres and their application for disease diagnosis via the polymerase chain reaction (PCR)(Montana State University - Bozeman, College of Agriculture, 1990) Baltazar, Baltazar MontesPolymerase Chain Reaction (PCR) protocols were developed for the diagnosis of net and spot forms of Pvrenophora teres. Low copy number sequences selected from a P. teres f. sp. maculata random genomic library were used as a source of probes. Emphasis was placed on those sequences identifying DNA polymorphisms between net and spot isolates and with little or no sequence similarity with barley, wheat, or triticale genomes. Sequences identifying a large deletion in genomic DNAs of net and spot isolates were preferred over sequences detecting small DNA changes. Sequence data of two informative clones, pPtm-290, and pPtm-60, were used to construct primer sets to amplify the corresponding sequence in genomic DNAs of net and spot isolates present in barley plants infected with these pathogens. PCR results demonstrated the potential of the PCR as a diagnostic tool for P. teres. All the PCR experiments conducted with primers designated as Pt-1 and Pt-2 constructed using the sequence data from pPtm-290, showed a strict correlation between the presence of a 430 bp band and the presence of the pathogen in genomic DNAs of barley infected with the net form, spot form or both pathogens. PCR experiments with primers Pt-3 and Pt-4 constructed using sequence data from pPtm-60, indicated that it is possible to detect polymorphic bands between net and spot isolates as evidenced by the PCR products analyzed in an ethidium bromide agarose gel. PCR analysis offers a sensitive, rapid, inexpensive, and non-radioactive technique for the diagnosis of P. teres infection in field-grown barley plants. Future experiments should focus on the ability of the PCR to detect P. teres and P. graminea in infected barley seeds. Additionally, PCR-based protocols for P. teres diagnosis could possibly be incorporated in seed certification programs to avoid the distribution of infected seed in farmer fields.Item Marker-assisted backcross breeding in wheat(Montana State University - Bozeman, College of Agriculture, 1996) Erpelding, John Edward