Scholarship & Research
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Item Development and characterization of a novel isothermal DNA amplification reaction(Montana State University - Bozeman, College of Engineering, 2021) Ozay, Burcu; Chairperson, Graduate Committee: Scott McCalla; This is a manuscript style paper that includes co-authored chapters.Isothermal nucleic acid amplification chemistries are gaining popularity as nucleic acid detection tools that can replace the current gold standard methods, PCR and its derivatives, with their simplicity, speed and applicability to point-of-care applications. In this work, we have developed and characterized a novel isothermal amplification chemistry, ultrasensitive DNA amplification reaction (UDAR). UDAR differs from similar chemistries with its unique, biphasic response with a high-gain output that can be captured with a cell-phone camera. The switch-like, nonlinear characteristics provide a definitive on/off signal for potential use in applications such as molecular diagnostics and DNA circuits. Tunability of the reaction was explored and the relationship between thermodynamic properties of the reaction templates and the reaction output was established. Limitations on fluorescent staining of reaction components by two popular commercial nucleic acid stains, SYBR Green II and SYBR Gold, were determined for a more accurate evaluation of the reaction output and reaction product analysis. A mathematical model of the reaction output was built and outputs from three different UDAR templates were successfully simulated. This model revealed important information on reaction pathways and helped identify the impact of individual reaction events. A comprehensive literature review of enhancement strategies for isothermal amplification reactions was conducted to serve as a guide to improve and modify these reactions according to different needs and applications. Lastly, UDAR was applied to microRNA detection, which are putative biomarkers for diseases such as cancer, malaria, and traumatic brain injury. Five different miRNAs were successfully detected by UDAR, down to 10 fM concentration. UDAR-based miRNA quantification is possible, with linear calibration curves provided between 10fM and 1 nM. This work has significant contributions to the growing field of isothermal nucleic acid amplification based-molecular detection systems by introducing a unique isothermal amplification chemistry, establishing design and manipulation techniques, and guiding improvement efforts of these technologies.Item Transduction of antigens into amplifiable DNA signals using structure switching aptamers(Montana State University - Bozeman, College of Engineering, 2019) Kayalar, Canberk; Chairperson, Graduate Committee: Stephanie McCallaDetection of specific antigens has one vital step in common: detection of biomarkers. Diagnostic testing that is rapid and reliable is unavailable in limited resource and rural settings. The solution to this need must be simple, inexpensive, robust, rapid and not require highly trained personnel to operate. Aptamers are capable of delivering those needs when matched with a novel high gain amplification method. This thesis focuses on important aspects of a novel protein detection assay that uses aptamers. Aspects that play an important role on the assay's success were investigated; aptamer selection and design of structure switching aptamers, designing DNA templates that will transduce the signal created by the aptamers, solid phase selection, aptamer immobilization on the solid phase, protein capture, and amplification of the signal. The first step was to find aptamers that were proven to specifically target clinically relevant targets and modify them to suit the needs of the assay. It is important to validate the aptamers' performance. The second important step was finding a solid phase that is compatible with the novel nucleotide amplification reaction that will be used to amplify the signal produced by the aptamers. Paramagnetic microbeads, membranes and polyacrylamide hydrogels were potential candidates for solid phases. Non-specific interaction of the target protein with the solid phase surface will not have negative effects while running the assay due to the structure switching of the aptamers however, it prevented the accurate quantification of the protein capture by aptamers. There is a need for the development of a blocking buffer that is specific to the solid phase. Washing of the excess DNA templates that are not bound to target-bound aptamers plays an important role in the assay's accuracy. The results presented here show the preliminary work that has been done for the novel protein detection assay that uses structure switching aptamers. This assay has the potential to detect diseases at point-of-care in low resource settings.