Sensitive Detection of Nucleic Acids with Bead-Based Microarrays

Nucleic acid sequences and mutations can be detected to differentiate species of bacteria, strains of viruses, or humans. Detailed study of genomes through the genotyping of single nucleotide polymorphisms (SNPs) is helping our understanding about heritable genetic diseases, drug resistance in microbes, and susceptibility to various forms of cancer. It is only a matter of time until the costs of next generation sequencing are sufficiently low enough to use whole- or partial-genome sequencing as a commonplace technique. Until then, methods that focus on SNPs or short sequences contained in genes will continue to be of high interest for diagnostic platforms.;The rapid detection of microbial contamination in food and water supplies is a high priority for public safety. Bacteria, viruses, and protozoa are often introduced into water supplies through fecal shedding by asymptomatic livestock and wildlife. This compromised water is then used by agricultural irrigation systems or consumed directly in the form of tainted drinking water. In the first major section of this thesis, I describe a multiplexed array with oligonucleotide probes coupled to microspheres, or beads, designed to detect virulence genes for many of the bacterial species associated with food and water contamination. The system was validated with stool samples spiked with several strains and species of Escherichia coli and Shigella. Water presents a technical challenge of how to detect these highly pathogenic organisms in large sample volumes. We developed a protocol using a continuous flow centrifuge (CFC) to concentrate bacteria from several liters of water. Microarray results are reported, along with a low-cost colorimetric dip test.;The second major section of this thesis describes work carried out with human samples for saliva diagnostics and maternal ancestry studies. The simultaneous detection of proteins and nucleic acid targets in saliva is of interest for non-invasive clinical testing. We developed methods to probe DNA and proteins from pathogenic bacteria that cause inflammatory responses in upper respiratory tract infections. For these methods we use an isothermal DNA amplification technique coupled with antibody-based protein detection to probe cytokines associated with the bacteria. Techniques for high throughput whole genome genotyping of nuclear DNA have been previously established, but currently are very expensive. Mitochondrial DNA (mtDNA) is an interesting counterpart to nuclear DNA, but mtDNA is a challenging genome to study due to its high number of repeated sequences and the density of mutations. The mitochondrial genome is maternally inherited and has a high rate of mutation, the pattern of which is associated with the maternal lineage of the spread of modern Homo sapiens. We designed and validated a genotyping assay to probe tens of mutations correlated with maternal ancestry. The results of this assay have fostered several undergraduate projects to develop low-cost readout methods suitable for high school and undergraduate teaching labs.;Platform development is described in the final chapter to frame the Walt group's transition from bead-based arrays to focusing on single molecule detection. The wells of etched fiber optic arrays were directly functionalized with capture probes. After capture, the wells were sealed to form reaction vessels capable of retaining the products of enzymatic turnover. In very dilute solutions, where the distribution of targets per well is either zero or one, the number of active wells can be counted to give a binary concentration readout. Future work based on this platform will enable studies about the differences in single cell mechanisms responsible for genetic recombination, cancer, and metabolic pathways. Understanding these processes at the single cell level will help spur the transition to medicine on the individualized level.;Two appendices are included, detailing early progress on the saliva diagnostics project, and a tutorial designed to explain the workflow of designing and validating oligonucleotide sequences for PCR amplification and microarrays.