PhD Dissertation Defense: "Developing an Ultrasensitive DNA Amplification Reaction", by Burcu Ozay
- Tuesday, July 27, 2021 from 11:00am to 12:00pm
- Via WebEx: https://montana.webex.com/meet/r17s481
DEVELOPMENT AND CHARACTERIZATION OF A NOVEL ISOTHERMAL DNA AMPLIFICATION REACTION
PhD Defense By Burcu Ozay, Department of Chemical & Biological Engineering, Montana State University
Tuesday, July27 ~ 11am-12pm
Via WebEx: https://montana.webex.com/meet/r17s481
Isothermal nucleic acid amplification chemistries are gaining popularity as nucleic acid detection tools that can replace the current gold standard method, PCR and its derivatives, with their simplicity, speed and applicability to point-of-care applications. In this work, we have developed and characterized a novel isothermal amplification chemistry, ultrasensitive DNA amplification reaction (UDAR). UDAR differs from similar chemistries with its unique, biphasic response, resembling Hill-type kinetics that is suitable for DNA-based switch systems and high-gain output that can be captured with a cell-phone camera. Tunability of the reaction was explored and the relationship between thermodynamic properties of the reaction templates and the reaction output was established. Limitations on fluorescent staining of reaction components by two popular commercial nucleic acid stains, SYBR Green II and SYBR Gold, were determined for a more accurate evaluation of the reaction output and reaction product analysis. A mathematical model of the reaction output was established and outputs from three different UDAR templates were successfully simulated. This model revealed important information on reaction pathways and helped identify the impact of individual reaction events. A comprehensive literature review of enhancement strategies for isothermal amplification reactions was conducted to serve as a guide to improve and modify these reactions according to different needs and applications. Lastly, UDAR was applied to microRNA detection, which are considered as biomarkers for diseases such as cancer, malaria, and traumatic brain injury. Five different miRNAs were successfully quantified by UDAR, down to 10 fM concentration. Sink mechanisms were explored where the none-specifically produced reaction products rendered unable to contribute to the signal. A switch mechanism was also established, with controlled shut-down of the signal for specific target concentrations.
This work has significant contributions to the growing field of isothermal nucleic acid amplification based-molecular detection systems by introducing a unique isothermal amplification chemistry, establishing design and manipulation techniques, and guiding improvement efforts of these technologies.
- Department of Chemical and Biological Engineering