Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2002 The ligase detection reaction: the evolution of a mutation detection strategy Hannah D. Farquar Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Chemistry Commons Recommended Citation Farquar, Hannah D., "The ligase detection reaction: the evolution of a mutation detection strategy" (2002). LSU Doctoral Dissertations. 3855. https://digitalcommons.lsu.edu/gradschool_dissertations/3855 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THE LIGASE DETECTION REACTION: THE EVOLUTION OF A MUTATION DETECTION STRATEGY A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College In partial fulfillment of the Requirements for the degree of Doctor of Philosophy In The Department of Chemistry By Hannah D. Farquar B.S., Louisiana Tech University, 1996 December 2002 Acknowledgements I would like to thank Dr. Robert P. Hammer for being patient enough to deal with my very steep learning curve and being an understanding colleague at the top of that curve. I would also like to thank Dr. Steven A. Soper for all of his teaching and support which has made me not only more successful but also a much better scientist. Dr. Kathleen Morden deserves much thanks for being a strong female scientist and a great role model. Dr. Tod J. Miller, a postdoctoral researcher, has had an enormous impact on my life as a graduate student. He was an academic mentor as well as a great friend to me. He taught me how to think through problems yet never lose the big picture. This lesson has proven to be one of great importance as I have dug deeper into the research hole. Dr. Andrea S. Saurage was incredibly helpful in my first years as a graduate student teaching me every technique I needed to begin my graduate studies and continues to be an immense support sharing her experiences post-LSU. I would also not have attained the level of satisfaction with my graduate studies had it not been for two very impressive undergraduate researchers with whom I have worked: Camille E. Tallini and Arsham Sheybani. They are not only two of the most intelligent people I have ever had the pleasure of meeting, but they are also two of the most open minded and well meaning human beings I believe I will ever come across. They are certain to be incredibly successful in every endeavor they attempt. Finally, I must thank my husband, George R. Farquar, for being so immensely wonderful and understanding. George, you not only give me the best scientific ii discussions and wonderfully patient answers to my not so well thought out questions, but you give me the security and sanity that is required to achieve the title of Ph.D. I can only hope that you understand how much of this dissertation as well as my life belong to you. iii Table of Contents Acknowledgments ......................................................................................................... ii List of Tables ................................................................................................................ vii List of Figures.............................................................................................................. viii List of Schemes............................................................................................................ xiv List of Abbreviations ...................................................................................................xv Abstract........................................................................................................................xix Chapter 1. Introduction.................................................................................................1 1.1 Mutations.......................................................................................................1 1.2 PCR ................................................................................................................6 1.3 DNA Polymerases..........................................................................................9 1.4 Mutation Detection .....................................................................................15 1.4.1 Scanning Methods.....................................................................16 1.4.2 Diagnostic Methods...................................................................18 1.5 References....................................................................................................24 Chapter 2. Nucleoside Base Analogs ..........................................................................29 2.1 Introduction.................................................................................................29 2.1.1 DNA Structure and Stability....................................................29 2.1.2 DNA Polymerase Recognition..................................................36 2.1.3 Nucleoside Base Analogs-The Literature................................37 2.1.3.1 DNA Stability ................................................................38 2.1.3.2 DNA Polymerase Recognition......................................43 2.2 Pyrazole-Based Analogs .............................................................................46 2.2.1 Introduction...............................................................................46 2.2.2 Synthesis.....................................................................................50 2.2.3 Computational Analysis...........................................................54 2.2.4 DNA Polymerase Recognition of Pyrazole Analogs...............60 2.3 Thiazole and Thiazole N-oxide Analogs....................................................63 2.3.1 Synthesis.....................................................................................64 2.3.2 Computational Analysis...........................................................70 2.3.3 Thermodynamic Analysis.........................................................76 2.3.4 DNA Polymerase Recognition..................................................78 2.4 Conclusions..................................................................................................84 2.5 References....................................................................................................85 iv Chapter 3. Nucleoside Backbone Analogs .................................................................92 3.1 Introduction ..............................................................................................92 3.1.1 Backbone Analogs-The Literature ..........................................93 3.1.2 Synthesis.....................................................................................96 3.2 Exonuclease Resistance of Phosphoramidate.......................................100 3.3 Conclusions..............................................................................................106 3.4 References................................................................................................106 Chapter 4. Capillary and Microdevice Electrophoresis of LDR ...........................110 4.1 Introduction...............................................................................................110 4.1.1 LDR of Colorectal Cancer......................................................110 4.1.2 Capillary and Microdevice Electrophoresis .........................112 4.2 Capillary Electrophoresis: Model Studies and LDR Reactions ..........114 4.2.1 Introduction.............................................................................114 4.2.2 Model Studies..........................................................................115 4.2.3 LDR Reactions........................................................................116 4.3 Microdevice Electrophoresis: Model Studies ........................................122 4.4 Conclusions................................................................................................125 4.5 References..................................................................................................125 Chapter 5. Experimental Details ..............................................................................129 5.1 Nucleoside Base Analogs-Chapter 2........................................................129 5.1.1 Synthesis of 1-(β-D-2´-deoxyribofuranosyl)-4-(2-thiazolyl)- pyrazole....................................................................................129 5.1.2 Computational Analysis of Pyrazole-Based Analogs...........130 5.1.3 DNA Polymerase Extension Reactions of Pyrazole Analogs.....................................................................................130 5.1.4 Synthesis of Thiazole and Thiazole N-oxide Analogs ..........131 5.1.4.1 Synthesis of 2-deoxy-3,5-di-O-p-toluoyl-β-D-erythro- pentofuranosyl cyanide (2).....................................................131 5.1.4.2 Synthesis of 2,5-anhydro-3-deoxy-4,6-di-O-p-toluoyl- D ribo-hexonthioamide (3) ......................................................132 5.1.4.3 Synthesis of 2-(2´-Deoxy-3´,5´-di-O-p-toluoyl-β-D- ribofuranosyl)thiazole (4).......................................................132 5.1.4.4