M.Sc Thesis

M.Sc StudentKhalandovsky Rebecca
SubjectHighly Specific Recognition of Nucleic Acid Sequences for
Point-of-Care Diagnostics Using Selectively
Focusing Probes and Isotachophoresis
DepartmentDepartment of Mechanical Engineering
Supervisor ASSOCIATE PROF. Moran Bercovici
Full Thesis textFull thesis text - English Version


Point-of-care diagnostics, which aim to diagnose a disease or condition quickly and without the use of laboratory equipment, require assays that are rapid, sensitive, specific, and onot labor-intensive. Recently, Ostromohov et al. developed the isotachophoresis with non-focusing probes assay (ITP-NFP), which is able to detect nucleic acids with a sensitivity of 100 fM; however, the specificity of this assay has not yet been studied. In this work, we derive the expected specificity and signal to noise ratio (SNR) of the assay in the presence of mismatch and randomly binding non-target sequences under the assumption of detection at steady state, and propose experimental conditions (temperature, and probe concentration, length, and type) that would optimize the specificity of the assay for different detection requirements.

We experimentally characterize the specificity of the existing assay at different temperatures, using different probe concentrations, lengths, and types. We demonstrate single-nucleotide specificity achieved by PNA probes at 55oC with applicability to 16S RNA detection for bacterial identification in urinary tract infection diagnosis, and show the potential applicability of similar PNA probes to detecting PML-RARA fusion transcripts in the diagnosis of acute promyelocytic leukemia (APL). We characterize the specificity of morpholino probes, which have not previously been used in ITP-based assays, and demonstrate increased discrimination of 1-5 nucleotide mismatches when using morpholino probes 14 nucleotides rather than 25 nucleotides long. The ability to use morpholino probes contributes significantly to the feasibility of using the ITP-NFP assay in a point-of-care setting since morpholinos are more soluble and easier to work with than PNA. Finally, we present a new model addressing the dissociation of non-specific bonds during the operation of the ITP-NFP assay, yielding non-steady-state kinetics that may contribute significantly to the signal to noise ratio obtained in the assay and that can be leveraged to further improve its specificity.