Detection of Single Nucleotide Polymorphisms in Resource-Constrained Settings

Abstract

Nearly half of known genetically linked human disorders are caused by single base variations, often referred to as single nucleotide polymorphisms (SNPs). Therefore, SNPs serve as a molecular biomarker which, when detected, can provide invaluable insights into disease susceptibility, progression, and treatment responses. Beyond the human genome, SNPs in pathogens, such as HIV-1, SARS-CoV-2, and Mycobacterium Tuberculosis, indicate important traits like drug resistance, transmissibility, and vaccine efficacy. Advancements in biotechnology and science have created powerful genetic tools for the identification of SNPs and other genetic variations. Yet, gold-standard technologies remain expensive and complex, making them unfit for application in resource-constrained settings. This dissertation focuses on innovative approaches to SNP detection that prioritize accuracy, affordability, accessibility, and adaptability. The oligonucleotide ligation assay (OLA), a high-fidelity, ligase-facilitated reaction, was employed for highly specific detection of known SNPs. The OLA was coupled with polymerase chain reaction (PCR) to attain robust and sensitive detection of important SNPs for pathogenic traits and human diseases. During the COVID-19 pandemic, my work focused on SARS-CoV-2 variant-typing to achieve sequencer-free identification of locally circulating variants of concern in clinical human-derived samples. While the variant-typing efforts were performed in a quick, high-throughput, and adaptable manner, some technical burden of assay implementation remained. Magnetic bead processing was integrated into the OLA-PCR coupled workflow to address this burden within the context of HIV-1 drug resistance detection, significantly improving assay sensitivity and provided the foundation for future automation. To further enhance accessibility, OLA and PCR assay design challenges were overcome through development of a software tool for automated nucleic acid reagent design. The tool streamlined assay customization for a variety of genetic targets, including M. Tuberculosis drug resistance mutations and SNPs indicative of disease risk. By innovating upon and around existing molecular technologies to overcome technical, logistical, and financial barriers, the work expands the reach of the OLA for highly specific SNP testing – improving the practicality of the assay for resource-constrained environments and situations. With this dissertation, efforts were focused toward not only advancing the field of genetic diagnostics but also toward providing a foundation for more equitable healthcare solutions worldwide.