Date of Award

5-2020

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

Nina Goodey

Committee Member

Ulrich Gubler

Committee Member

Kirsten Monsen-Collar

Abstract

A key problem when developing molecular diagnostic techniques is procuring target detection probes. The need of a stable and selective probe is necessary for accurate target identification in disease detection. Antibodies are widely used but they are unstable at ambient temperatures and are expensive and time consuming to develop. The use of aptamers (short singlestranded DNA) as molecular probes provides several advantages over protein-based methods because they are more stable, easily selected and modifiable. The need of a reliable and stable probe is necessary for accurate detection tests in the field for disease monitoring. Currently, field methods are limited in the time and expertise required to obtain results. Our objective is to enrich a starting library of aptamers (~10₁₄ randomized sequences) to be specific towards Ranavirus through SELEX methods. We describe a dual SELEX approach using two Ranavirus targets, whole Ranavirus FV3 virion particles and its MCP. After 6 rounds of stringent conditions followed by a cross-reaction selection with virion specific aptamers and the MCP protein target, we have successfully enriched a starting library of 10₁₄ non-specific aptamers to those specific to Ranavirus. Aptamer sequences were analyzed by Sanger and Next-Generation Sequencing methods for common structural motifs between virion specific aptamers and MCP specific aptamers, to demonstrate enrichment of the starting library. In future work, the binding affinities of selected aptamer probes will be measured by either electrophoretic mobility shift assays (EMSA) or Surface Plasma Resonance (SPR) using the MCP target. We expect binding affinities of aptamers to be within the nanomolar range, demonstrating the aptamers high affinity and specificity. Ultimately, the selected aptamers will be incorporated as molecular probes for Ranavirus into a field usable, rapid pathogen test that will surpass the limitations of current field tests.

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