Tuning DNA Stability to Achieve Isothermal DNA Amplification

Abu Kausar
Developing a simple, general, and isothermal self-replicating system is one of the key requirements for a simplified detection platform for nucleic acid sequences specific to a disease causing microrganism. One of the challenges in achieving nucleic-acid templated isothermal amplification is that the product duplex formed after the templated reaction is much more stable than the prereaction complex. Consequently, the template remains bound to the complementary nucleic acid formed, preventing
more » ... template from initiating another cycle of amplification. To avoid this product inhibition, we introduce different destabilizing linkers in the template, which result in the isothermal amplification of complementary DNA. One of the most active destabilizing groups is an abasic lesion, which we demonstrate can also operate in acrosscatalytic process that we refer to as lesion-induced DNA amplification or LIDA. Using LIDA, we are able to observe rapid, isothermal self-replication of DNA, of a variety of sequences, by combining two cycles of amplification and using a high concentration of T4 DNA ligase enzyme. Moreover, we find the LIDA process operates in the presence of genomic DNA and can detect a specific sequence in plasmid DNA, suggesting this method may find application in nucleic-acid based diagnostics. Additionally, as DNA ligases have a better discriminating ability than DNA polymerases for mismatches in target DNA, using ligase-based LIDA we are able to achieve very good discrimination between amplification initiated with a matched target versus a mismatched target (discrimination ratio = 47). iii Combining a simple amplification method for disease-specific nucleic acid sequences with a simple detection platform is an important step towards the development of a diagnostic tool usable in resource-limited environments. Traditionally, we employ polyacrylamide gel electrophoreses (PAGE) to detect the amplified nucleic acid, requiring that we label one of the DNA probe strands that is incorporated into the self-replication product. In another approach explored, one of the probes is modified with a fluorescent donor (FAM) and another probe modified with a fluorescent acceptor (Cy5). The replication process causes these probes to become covalently attached, which we observe by Förster Resonance Energy Transfer (FRET) providing a real-time method of detection of the amplification process. To further simplify detection, we also combine LIDA with a rapid colorimetric approach developed in our group based on gold nanoparticles, where the target-induced amplification product leads to the disassembly of nanoparticle aggregates resulting in a color change from purple to red. Additionally, we have screened different DNA ligases and demonstrate that we are able to detect down to 14 pM target DNA using E. coli DNA ligase (compared with 1.4 nM using T4 DNA ligase), which suggests that identifying or developing an enzyme with no blunt-end ligation would be ideal for further lowering the limit of detection with LIDA. Finally, varying the base across from the abasic site reveals that there is no special base requirement for the isothermal system to work, providing further evidence that LIDA represents a general approach for isothermal target sequence amplification. iv Preface This project on isothermal DNA amplification by destabilization was highly collaborative. The idea in Chapter 2 was conceived by J. M. Gibbs-Davis. Prior to my joining the project, some of the proof-of-concept experiments were done by J. Lam and R. D. McKay. R. Bhogal and A. Tang synthesized several of the destabilizing templates reported in Chapter 2 and performed some of the thermal denaturation curves reported therein. J. Lam synthesized some of the DNA strands and performed some of the single cycle ligation and amplification experiments as well as some of the thermal denaturation curves in that chapter. The data reported in this chapter for the kinetic experiments on the single cycle ligations and the cross-catalytic amplification was studied by myself. Chapter 2 has been published as
doi:10.7939/r3xg9fg80 fatcat:6vvcft2ebvdhxlolel73gggxly