Isothermal Amplification Techniques for the Detection of Nucleic Acids and Proteins

Abstract

The early detection of disease is beneficial for improved patient prognoses. One major challenge in early disease detection is the small, undetectable amounts of biomarkers. Detection strategies that confer amplification of the biomarker itself or of the detection signal are therefore desirable. Furthermore, emerging strategies conferring amplification at isothermal temperatures offer improvements in technical procedures by circumventing the requirement for multiple different reaction temperatures as is required in polymerase chain reaction. Despite the advances in isothermal amplification strategies using nucleic acids, there are still some drawbacks such as the technical difficulty of protocols and number of reagents required. The primary objective of my research was to develop novel techniques to improve and simplify the detection of nucleic acid and protein targets. In order to address this objective, I developed three new techniques for the isothermal and amplified detection of nucleic acid and protein targets with improved features. To improve isothermal and exponential amplified detection, I developed a new technique called Beacon-mediated Exponential Amplification Reaction (BEAR) to detect nucleic acid targets. BEAR only required a single enzyme and a single primer. I applied BEAR to detect Myoclonus Epilepsy with Ragged Red Fibres (MERRF). I achieved a limit of detection of 10 fM in 80 min and a recovery of ~91% in cell lysate for the MERRF sequence using BEAR. To improve isothermal and amplified detection without using enzymes, I developed a new turn-on fluorescence technique inspired by hybridization chain reaction (HCR) enabling the generation of turn-on fluorescence signals from label-free hairpins. This HCR technique uses four hairpins which overcomes the background that could arise when using two label-free hairpins. Using this new technique, I achieved a limit of detection of 660 pM of a nucleic acid target in solution when using 50 nM of hairpins in 30 min at room temperature. To improve the protocols of localized protein imaging, I adapted the concept of bindinginduced DNA assembly (BINDA) to the developed HCR technique. I used BINDA to convert protein binding into the generation of a DNA strand. The formation of the DNA strand initiated the HCR that produced fluorescence signals. I applied this technique to detect a HER2+ breast cancer cell line where membrane fluorescence indicating the HER2 status of the cells was achieved in as soon as 5 min with strong fluorescence signals at about 45 min to 60 min. This technique did not require any enzymes or washing steps and was performed at room temperature. These developed techniques feature isothermal reaction temperatures, low reaction volumes, and technically simple protocols because they are all in mix-and-read formats. These features allow potential applications of my techniques for improved clinical laboratory testing, point-of-care assays, and testing in resource-limited settings. Furthermore, the modularity of these developed DNA designs allows for the adaptation to other targets as well.