Rapidly diagnosing the precise drug resistance present in sepsis-inducing bacteria is a continuing need to maintain the efficacy of our medical systems. Diagnostics currently being developed for such scenarios are either sensitive or rapid, but not both. Sequence-specific single DNA molecule analysis could fill this gap if it could be adapted to work on smaller targets, similar to those produced by classical biological methods. In this work, I demonstrate that immobilized ssDNA in the appropriate hybridization buffer can rapidly pull its complementary sequence out of solution. I also demonstrate that such systems in a microfluidic chip can be used to capture bacterial plasmids as a step toward subsequent multiplexed analysis. Finally, I demonstrate that a 120 bp double stranded polynucleotide with an overhanging single stranded 25 bp probe sequence can be modified with multiple fluorophores and used to label captured targets in a sequence-specific manner. This system shows that it is possible to label bacterial plasmids in a manner that can bridge the technological gap between single molecule counting and small oligonucleotide targets. Such a system can achieve lower limits of detection for clinically relevant samples while maintaining rapid processing times.
College and Department
Chemistry and Biochemistry
BYU ScholarsArchive Citation
Hanson, Robert L., "Sequence Specific Concentration and Labeling of Bacterial Plasmids for Future Use in Detection of Drug-Resistant Sepsis Cases Without Amplification" (2021). Theses and Dissertations. 9033.
Microfluidics, DNA, hybridization, sepsis, lab on a chip, drug resistance