ISOTHERMAL DNA DETECTION UTILIZING BICYCLIC AMPLIFICATION OF PADLOCK PROBES

Abstract

As healthcare worldwide changes to more patient-centric models, medical diagnostics need to adapt to being used in settings outside of the central lab. Current strategies to bring diagnostics to the patient’s bedside involve miniaturizing complicated amplification techniques, such as polymerase chain reaction, or building convoluted microfluidic assays that are difficult to operate. Ideally, a patient-centric diagnostic would require little instrumentation or training to operate, for which isothermal amplification techniques are ideal. Recent developments in catalytic DNA have enabled novel ways of iterating on amplification strategies to detect medically-relevant target sequences in systems that require little manipulation to operate. In this thesis we improve upon the body of research on DNAzymes, catalytic DNAs that can self-cleave in the presence of a cofactor, used in concert with amplification techniques. We create a one-pot, bicyclic amplification assay capable of detecting single-stranded oligonucleotides, with straightforward extensions to double-stranded targets, multiplexing, and integration into advanced detection platforms. The target is detected through its hybridization to a circle template, using the sequence specificity of DNA to splint the ligation of this ‘Template I,’ with minimal detection of off-target sequences. The circular Template I is copied through rolling circle amplification (RCA), with the amplicon containing a DNAzyme that will self-cleave in the presence of copper ions. This generates a second primer in situ that can be used to prime a second, pre-ligated, Template II to elevate the RCA amplification scheme from a linear method to a polynomial one. This Circle II template can then be used in a variety of detection modalities. The second amplicon can be used to cleave a hybridized FRET probe through the same copper ion cleavage mechanism as the primer generation, resulting in real-time fluorescence tracking. Alternatively, the RCA of the second circle can produce G-quadruplexes, which can be visualized with ABTS as a colorimetric endpoint that can be seen by eye, reducing the need for peripheral electronics. Finally, this thesis demonstrates the performance of the bicyclic RCA system in a phase-change system providing sequential mixing of components separated by wax layers, allowing the assay to proceed without any user interaction other than heating.