Browsing by Author "Ozay, Burcu"

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Development and characterization of a novel isothermal DNA amplification reaction
(Montana State University - Bozeman, College of Engineering, 2021) Ozay, Burcu; Chairperson, Graduate Committee: Scott McCalla; This is a manuscript style paper that includes co-authored chapters.
Isothermal nucleic acid amplification chemistries are gaining popularity as nucleic acid detection tools that can replace the current gold standard methods, 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 with a high-gain output that can be captured with a cell-phone camera. The switch-like, nonlinear characteristics provide a definitive on/off signal for potential use in applications such as molecular diagnostics and DNA circuits. 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 built 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 putative biomarkers for diseases such as cancer, malaria, and traumatic brain injury. Five different miRNAs were successfully detected by UDAR, down to 10 fM concentration. UDAR-based miRNA quantification is possible, with linear calibration curves provided between 10fM and 1 nM. 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.
First characterization of a biphasic, switch-like DNA amplification
(2018-03) Ozay, Burcu; Robertus, Cara M.; Negri, Jackson L.; McCalla, Stephanie E.
We report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1 pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.
A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions
(2021-11) Ozay, Burcu; McCalla, Stephanie E.
Isothermal nucleic acid amplification techniques are used to detect a variety of molecules such as RNAs, DNAs and proteins. When compared to the gold standard polymerase chain reaction, isothermal methods are rapid and simple but often have lower specificity and sensitivity. In this review, we provide a summary of reaction additives and techniques that enhance the sensitivity, specificity, stability, robustness, or speed of isothermal nucleic acid amplification reactions used for sensing applications. We also provide the mechanism and purpose of each enhancement technique, which can provide guidance when selecting reaction additives to optimize novel isothermal amplification systems and assays.