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Development of Potential Strategies for DNA Quantification and DNA Probes for Detection of Legionella Pneumophila

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Development of Potential Strategies for DNA Quantification and DNA Probes for Detection of Legionella Pneumophila Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 Development of potential strategies for DNA quantification and DNA probes for detection of Legionella pneumophila Parker James Dulin Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Civil Engineering Commons, and the Environmental Engineering Commons Recommended Citation Dulin, Parker James, "Development of potential strategies for DNA quantification and DNA probes for detection of Legionella pneumophila" (2019). Graduate Theses and Dissertations. 17673. https://lib.dr.iastate.edu/etd/17673 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Development of potential strategies for DNA quantification and DNA probes for detection of Legionella pneumophila by Parker James Dulin A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Civil Engineering (Environmental Engineering) Program of Study Committee: Kaoru Ikuma, Co-major Professor Carmen Gomes, Co-major Professor Chris Rehmann The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Parker James Dulin, 2019. All rights reserved. ii TABLE OF CONTENTS Page LIST OF FIGURES ....................................................................................................................... iv LIST OF TABLES ......................................................................................................................... vi NOMENCLATURE ..................................................................................................................... vii ACKNOWLEDGMENTS ........................................................................................................... viii ABSTRACT ................................................................................................................................... ix CHAPTER 1. GENERAL INTRODUCTION ................................................................................1 Methods of Detection for Waterborne Pathogens ..................................................................... 2 Opportunistic Premise Plumbing Pathogens ............................................................................. 4 Biosensors .................................................................................................................................. 7 DNA Quantification ................................................................................................................ 13 DNA Hybridization ................................................................................................................. 16 Scope of Research ................................................................................................................... 18 References ............................................................................................................................... 19 CHAPTER 2. METHODS FOR ACCURATE QUANTIFICATION OF STRUCTURED DNA PROBES AND DOUBLE-STRANDED DNA AFTER HYBRIDIZATION USING THE QUBIT FLUOROMETER .............................................................................................................27 Abstract .................................................................................................................................... 27 Introduction ............................................................................................................................. 28 Procedures ............................................................................................................................... 31 Quantification of Structured DNA Probes ......................................................................... 32 Quantification of DNA Hybridization ................................................................................ 35 Additional Information and Method Validation ...................................................................... 36 Conclusions ............................................................................................................................. 42 References ............................................................................................................................... 43 CHAPTER 3. THE EFFECT OF STRUCTURE ON DNA HYBRIDIZATION IN SOLUTION AND IMMOBILIZED ON A PARTICLE SURFACE FOR OPTIMIZED DETECTION OF LEGIONELLA PNEUMOPHILA ...................................................................................................46 Abstract .................................................................................................................................... 46 Introduction ............................................................................................................................. 47 Materials and Methods ............................................................................................................ 49 Solution Study .................................................................................................................... 52 Particle Study ..................................................................................................................... 55 Statistical Analysis ............................................................................................................. 60 Results and Discussion ............................................................................................................ 60 Solution Study .................................................................................................................... 60 Characterization of DNA Conjugated Magnetic Particles and Immobilized Hybridization Conditions ................................................................................................... 65 iii Particle Study ..................................................................................................................... 66 Conclusions ............................................................................................................................. 68 References ............................................................................................................................... 69 CHAPTER 4. GENERAL CONCLUSIONS .................................................................................73 Engineering Significance ......................................................................................................... 74 Future Recommendations ........................................................................................................ 75 APPENDIX. PCR PROTOCOLS ..................................................................................................77 References ............................................................................................................................... 79 iv LIST OF FIGURES Page Figure 1.1. Reported cases of Legionnaires’ disease since the beginning of the 21st century. ....... 5 Figure 1.2. Diagram of the two major components of a biosensor. ................................................ 8 Figure 1.3. Example of a sandwich DNA hybridization strategy for biosensing detection............ 9 Figure 1.4. Schematic of a potential microfluidic device for an electrochemical DNA-based biosensor. .................................................................................................................. 12 Figure 1.5. Examples of DNA motifs that are available through intramolecular and intermolecular interactions. ...................................................................................... 17 Figure 2.1. Schematic of the process for quantification of structured DNA probes via denaturation with DMSO followed by fluorescent dye treatment. ........................... 31 Figure 2.2. Schematic of the process for quantification of DNA hybridization via enzymatic digestion followed by fluorescent dye treatment. ..................................................... 34 Figure 2.3. Comparison between ssDNA concentrations measured with Qubit and NanoDrop. ................................................................................................................ 37 Figure 2.4. Linear calibration for the determination of ssDNA concentration dissolved in DMSO. ...................................................................................................................... 39 Figure 2.5. Measured concentrations of dsDNA spiked with concentrations of ssDNA to assess the effect of ssDNA on background noise. .................................................... 40 Figure 2.6. Concentrations of dsDNA spiked with concentrations of ssDNA and treated with Exonuclease I.. ................................................................................................. 42 Figure 3.1. Linear calibration for the determination of ssDNA concentration dissolved in DMSO. .....................................................................................................................
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