SIMPLIFIED NUCLEIC ACID ISOLATION AND DETECTION TECHNIQUES FOR POINT-OF-CARE APPLICATIONS
Nanayakkara, Imaly Anoshka
White, Ian M
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The first step towards the treatment of any disease is diagnosis; however, a major hindrance towards monitoring diseases remains the availability of rapid diagnostic tests to detect DNA and RNA biomarkers.Unfortunately, traditional assays to test for these analytes are limited to centralized laboratories. For example, the gold-standard sample preparation methods to isolate and purify nucleic acid analytes require multiple hands-on steps, numerous chemicals, and specialized equipment. Recent research endeavors have made incremental progress to simplify these procedures by miniaturizing these steps, but still require the numerous chemicals and bulky peripheral instrumentation for operation. Similarly, gold-standard detection methods, namely quantitative PCR (qPCR), rapidly cycle between near-boiling temperatures, thus mandating the use of sophisticated instrumentation with high power demands. These methods for sample preparation and target detection are overall laborious, time-consuming, and require technical expertise thereby imposing large time, and financial costs for diagnoses. To address these challenges, this dissertation aims to shift the paradigm of how we approach sample preparation for nucleic acid isolation, and to design novel assays for rapid, specific, and multiplexed detection in a single reaction. First, a new sample preparation method is presented that simultaneously accomplishes three time-consuming sample preparation steps (cell lysis, DNA capture, and purification) in less than ten minutes. This platform enables DNA isolation from whole blood droplets, with subsequent amplification and detection of both genomic and pathogenic DNA bound to the microparticle surface. Second, we explore optimizations of a popular isothermal DNA amplification technique that previously demonstrated multiplex detection of bacterial genomes. By developing a triplex assay towards the identification of drug resistant bacteria, we address inhibition that is normally observed with this technique, and present strategies towards achieving amplification within 30 minutes. Lastly, we outline design consideration towards the development of a novel amplification scheme specifically for microRNA targets. This system leverages both DNA and RNA polymerases to achieve positive feedback and thus, requires evaluation of enzymes, sequence design, and buffers to inform assay design. Together, this work advances the development of NAATs towards a simplified, specific and multiplexed system.