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
Physical and Mathematical Sciences; 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