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Amplification of Nucleic Acids using Lesion-Induced DNA Amplification at Room-Temperature
- Author / Creator
- Alladin-Mustan, Bibi Safeenaz
The ability to amplify nucleic acid biomarkers at room temperature has remained elusive despite the great need of diagnostics suitable for the point of care. This thesis explores the probability of making lesion-induced DNA amplification (LIDA) an equipment-free platform that can work at room temperature. In LIDA, previous work in our group had shown a destabilizing lesion, specifically a model abasic group, could be used to achieve turnover and isothermal amplification. Therefore, to exponentially amplify DNA within a wide range of ambient temperatures (18-26 °C), we explored the addition of a second destabilizing group (a mismatch or another abasic group) in our isothermal lesion-induced DNA amplification system. We showed that we could tune the optimum temperature (To) at which the ligation reaction works best below 30 °C by the addition of a second destabilizing group to the system as this change results in further destabilization in LIDA. The magnitude of the destabilization is dependent on the type or mismatch or second abasic lesion present. Since this temperature range covers a wide range of room temperatures, we next demonstrate rapid DNA amplification at the bench without a heat source using a set of LIDA probes containing an A:C mismatch and an abasic site. These results show that the presence of a second lesion in the system makes the LIDA reaction faster at lower temperatures. However, when performed in one pot, that is in the presence of both the mismatched probe and the perfect probes, the more complementary system dominates the amplification process compared to the less complementary system.
RNA is another important biomarker for disease diagnosis. One challenge in point-of-care diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Therefore, our next goal was to show the versatility of LIDA towards RNA as a target. We performed a reverse transcription ligase chain reaction using our isothermal lesion induced DNA amplification technique that can be tuned to operate at any desired temperature. Using RNA-triggered LIDA, we can detect as little as ~100 attomoles of target RNA and can distinguish RNA target from total cellular RNA. Lastly, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/ detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.
Finally, I will discuss our efforts to decrease the background-triggered ligation reaction observed in LIDA. This background-triggered reaction occurs in the absence of a template and stems from the four probes used in LIDA. The four probes form a pseudo-blunt end, which is slowly ligated by T4 DNA ligase to generate the target template, triggering LIDA. T4 DNA ligase uses ATP as cofactor and is involved in all the three steps involved in the mechanism of ligation by the enzyme. The Hili group showed that modified ATP influences the specificity of the ligation reaction of nicked DNA duplexes. Therefore, we hypothesized that using ATP derivatives bearing different modifications, the pseudo-blunt end ligation might be perturbed to a much bigger extent compared to the DNA-templated reaction. We screened three ATP derivatives and one of them, 2-amino-ATP, gave better separation between the kinetic traces of LIDA initiated by template and no template at 1 mM 2-amino-ATP concentration. We found that ligation occurs only in the cycle that has the 5’-phosphate abasic and 3’-hydroxy adenosine, and that this pseudo blunt end ligation reaction is significantly decreased when the 2-amino-ATP and 6-methylamine-ATP were used.
In conclusion, this thesis explores the amplification of DNA and RNA at room temperature using lesion-induced DNA amplification as well as the effect of various ATP derivatives to reduce the background-triggered reaction observed in LIDA.
- Graduation date
- Fall 2020
- Type of Item
- Doctor of Philosophy
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