Browsing by Subject "PCR"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
Item Functionalization of Magnetic Nanoparticles with Deoxynucleotide Triphosphates for Utilization in DNA Sequencing(2023-02) Nazareth, CalvinCurrent methods for DNA sequencing leave a lot to be desired. There is immense research focused on developing methods to sequence longer and larger samples of DNA and to do so more rapidly. One possible new approach to DNA sequencing uses magnetically labeled deoxynucleotide triphosphates (dNTPs) as the complimentary bases for a sequencing-by-synthesis approach. The polymerase in this system would be immobilized on a sensor so that when the nucleotide is attached to the growing strand, the sensor can detect the signal. As the polymerase completes the strand for DNA extension, the signal from each magnetic nanoparticle (MNP) is recognized and generates a sequence.The contents of this thesis provide results for two critical initial steps in developing the proposed method. Firstly, we attached dNTPs to magnetic nanoparticles to create dNTP-MNP conjugates. We attempted two methods of conjugation with numerous characterization techniques to confirm the attachment. Secondly, we studied how the dNTP-MNP conjugation affected the dNTPs in quantitative polymerase chain reaction (qPCR) to model how they would interact with a polymerase in a hypothetical sequencer. The characterization results confirmed the conjugation of the dNTP to the MNP. qPCR was performed with various conditions and solvents which led to the conclusion that the MNP conjugation does not hinder polymerase interaction with the dNTP. This is a crucial result for the development of such a sequencer by providing the groundwork for the continuation of the project.Item Graft Copolymer Stabilized Gold Nanoparticles and Their Applications and Chemically Cleavable Linkers and Their Applications(2013-05) Kang, Jun SungThis thesis consists of two parts: (1) graft copolymer stabilized gold nanoparticles (AuNPs) and their biological application and (2) chemically cleavable 𝛼-azido ether and its biological application. In the first part, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymers that bear multiple thiol groups on the polymer backbone are used for exceptional ligands to stabilize AuNPs. To characterize the effect of copolymer structure on AuNP stability, we synthesized PLL-g-PEGs with different backbone lengths, PEG grafting densities, and number of thiols per polymer chain. AuNPs were then combined with these polymer ligands, and the stabilities of the resulting AuNP@PLL-g-PEG particles against high temperature, oxidants, and competing thiol ligands were characterized using dynamic light scattering (DLS), visible absorption spectroscopy, and fluorescence spectrophotometry. Our observations indicate that thiolated PLL-g-PEG ligands (PLL-g-[PEG:SH]) combine thermodynamic stabilization via multiple Au-S bonds and steric stabilization by PEG grafts, and the best graft copolymer ligands balance these two effects. This new ligand system enables AuNPs to be used for solid phase polymerase chain reaction (SP-PCR) that requires harsh reaction conditions, such as, elevated temperature and competing thiol molecules. Azide functionalized PLL-g-[PEG:SH] were conjugated to oligodeoxy-nucleotide (ODN) primers via click chemistry and bound to AuNPs to yield AuNP-primers that successfully primed target DNA synthesis on the surface of the AuNPs through PCR, as demonstrated by gel electrophoresis, DLS, and fluorescent analysis. Moreover, the graft copolymer stabilized AuNPs were applied to rapid DNA diagnostics in a single PCR tube with magnetic particles through color change without any instrumental analysis. In the second part, bioorthogonal, chemically cleavable 𝛼-azido ether has been studied and used to develop novel degradable materials. In order to understand the chemistry of the 𝛼-azido ether, model molecules bearing the 𝛼-azido ether were prepared. Hydrolytic stability of the model molecules was investigated by measuring their degradation rate using NMR, which leads to the relationship between the stability and chemical structures. Additionally, the cleavage kinetics of the model molecule, which was triggered by a couple of azide reducing reagents, was studied by NMR and UV-Vis absorption spectroscopy. The kinetic studies enable us to develop mechanistic investigation of the chemical cleavage as well as optimal cleavage conditions. Furthermore, the products after the chemical cleavage of the 𝛼-azido ether were characterized using NMR. The novel 𝛼-azido ether was then incorporated into degradable polyacrylamide gel electrophoresis (PAGE), in which biological macromolecules, including plasmid, microRNA, and proteins, were separated electrophoretically and recovered from the gel matrix with the optimal cleavage conditions. The kinetics of the recovery was quantitatively studied using UV-Vis absorption spectroscopy and fluorescence spectrophotometry. Furthermore, the recovered biological macromolecules were analyzed to investigate biocompatibility of our system. We anticipate further expansion of the 𝛼-azido ether to a broad range of biological applications based on the fundamental studies and the representative example in PAGE.Item A Rapid and Power-Efficient Laser-Induced Graphene Micro-Thermocycler for DNA Polymerase Chain Reaction(2023-06) Nguyen, KhoiBy amplifying millions or billions of copies of a DNA’s specific segment, polymerase chain reaction (PCR) has a wide range of applications in DNA cloning, forensics, medical, and infectious disease detection. As a result, the technology is becoming more and more widespread as the COVID-19 pandemic pushes for the need for rapid nucleic acid testing, and for early diagnosis compared to antigens testing. This research aims to develop a laser induced graphene (LIG) heater as the heating element for PCR point-of-care testing (POCT), as the PCR detection result is highly dependent on following a specific reagent heating cycle. In particular, the LIG heater can be integrated directly on a polymeric film by laser scribing in one step, eliminating the need for photolithography or chemical vapor deposition, and the entire device can be fabricated in under 5 minutes. This work investigates the heating performance of the LIG heater, by both Ansys Fluent simulation and actual experimentations. To efficiently control the thermal cycling, a LabVIEW program is constructed to obtain the voltage, temperature, and electrical current of the device, and it is highly adjustable to obtain the maximum heat ramp rate of 11.2 °C/s, and cooling rate of 10.8 °C/s in ambient condition. As a result, the thermocycler requires approximately 510 mW to maintain at 95 °C, with an accuracy of ±0.18 °C. Moreover, the device has shown high stability after 24 hours of repeated thermal cycling, with the average power consumption goes from 365 mW to 368 mW. With low power consumption and rapid fabrication, LIG shows great potential not only for PCR heating elements but also for chemical, gas sensors, flexible electronics, and various other applications.Item Understanding transmission of Leptospira at the household level through an examination of human behaviors, environmental water contamination, and animal carriers(2016-10) Mason, MeghanObjective. Leptospirosis is a zoonotic disease that contributes to human morbidity and mortality worldwide, and is the result of contact between mucous membranes or broken skin and the urine of infected animals either directly, or through contaminated soil or water. Rodents, livestock, and dogs are all able to shed the Leptospira bacteria in their urine. This research seeks to contribute to the understanding of transmission dynamics of the pathogen through an examination of human behaviors, pathogen diversity, and interactions between animal hosts and the environment. Methods. The data for the studies presented in this thesis come primarily from the Eco-epidemiology of Leptospirosis study conducted in the Los Rios Region, Chile from 2010-2012. The study collected survey data from 422 households in twelve different communities representing rural farm areas (4 communities), rural villages (4 communities), and urban slums (4 communities). At each household, survey data, environmental water samples, and blood samples from humans and animals were obtained. Rodents were also trapped and euthanized. Polymerase Chain Reaction (PCR) methods were used to detect Leptospira in rodents and water samples, while Microscopic Agglutination Testing (MAT) identified humans and animals with evidence of prior leptospirosis infection. The secY region of PCR-positive water samples was amplified and sequenced for phylogenetic analysis. Additional observational data was collected in 2013 from dogs in urban slums and was used to create an agent-based model for leptospirosis transmission in NetLogo version 5.0.3. Results. There were no discernable patterns of Leptospira diversity across the three community types studied (rural farm areas, rural villages, and urban slums), but the diversity of the pathogen did vary across the twelve individual communities. L. interrogans, L. kirschneri, and L. weilli were found in all community types (rural farms, rural villages, and urban slums), in descending order of frequency. There was no evidence that community-level prevalence of Leptospira in dogs, rodents, or livestock influenced diversity of the pathogen in the environment. According to agent-based modeling, both rodents and dogs contribute to environmental contamination and subsequent human infection in urban slum areas, but dog-based interventions (culling of stray dogs, and vaccination and restricting movement of owned dogs) are not expected to reduce human incidence of the disease. Addressing indirect transmission of Leptospira from the environment to humans was identified through the agent-based model as a better approach to reducing incidence. Most households in the study (95%) reported participating in some form of rodent control, but practices such as wearing boots or gloves when working with livestock or in the garden were less common. The proportion of households that participated in protective practices against zoonotic infection was heavily dependent on the community in which the households existed. Conclusions. The transmission and diversity of leptospirosis in endemic areas is driven by community-level factors. Efforts to reduce transmission through human engagement in protective practices should address the needs and norms of the individual communities. These studies have provided the framework for systematic surveillance and modeling strategies that can help to inform communities about potential contaminated environments, identify changes in the ecology of the bacteria that may signal an uptick in human infection, and suggest targeted interventions. As future research improves diagnostic tools, understanding of the survival of the pathogen in the environment, and shedding patterns by animal hosts, these proposed methods can be refined to better serve the needs of communities where this neglected tropical disease is endemic.