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Capillary and Microchip Gel Electrophoresis Using Multiplexed Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2005 Capillary and microchip gel electrophoresis using multiplexed fluorescence detection with both time- resolved and spectral-discrimination capabilities: applications in DNA sequencing using near- infrared fluorescence Li Zhu Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Chemistry Commons Recommended Citation Zhu, Li, "Capillary and microchip gel electrophoresis using multiplexed fluorescence detection with both time-resolved and spectral- discrimination capabilities: applications in DNA sequencing using near-infrared fluorescence" (2005). LSU Doctoral Dissertations. 107. https://digitalcommons.lsu.edu/gradschool_dissertations/107 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]. CAPILLARY AND MICROCHIP GEL ELECTROPHORESIS USING MULTIPLEXED FLUORESCENCE DETECTION WITH BOTH TIME-RESOLVED AND SPECTRAL-DISCRIMINATION CAPABILITIES: APPLICATIONS IN DNA SEQUENCING USING NEAR-INFRARED FLUORESCENCE 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 Li Zhu B.S., Nankai University, 1998 December, 2005 DEDICATION This dissertation would not be possible without the love and support of my family and it is to them that I dedicate this work. To my parents, Yunlin Zhu and Defen Han. You are the best of examples for your children. I can never thank you enough for what you have done for me. To Aimin Xu, my husband and best friend. Thank you for your constant love, understanding and encouragement. Your love and passion has always lifted me up and helped me keep perspective. To my sisters and brother, who have contributed so much to my life: Yinghong Zhu Ming Zhu Manwen Zhu ii ACKNOWLEDGEMENTS I would first like to express my gratitude to my major advisor, Dr. Steven A. Soper, for his inspiration, patience, teaching and mentoring. Your constant support and guidance contributed to every accomplishment presented here. You helped to mold me into the scientist I am today. I would like to acknowledge Drs. Robin L. McCarley, Robert M. Strongin, Isiah M. Warner and John R. Battista, who have participated as members of my committee. I sincerely appreciate your patience and time reading my dissertation and providing valuable advice and comments. I would like to acknowledge Dr. Wieslaw J. Stryjewski. Thank you for your contributions to my research. You are not only a research colleague but also a teacher and friend to me. Your intelligence and expertise really inspired me in the early years of my study. I would like to acknowledge Dr. Robin L. McCarley (again!) and Dr. Robert P. Hammer for the career assistance they gave me. Thank you for all your time and support. I would like to thank Dr. Zhidong Zhao for his contributions to the development of the scanner. I would also like to thank the helpers from our electronic and machine shops, particularly Mr. Marcus Nauman and Mr. Donnie Orlano. Thank you for all your assistance and discussions in developing the scanner. I would like to thank the entire Soper Research Group, both former and current members, who are too many to name. I sincerely cherish all the moments we shared together as colleagues and friends. I will miss you all. iii I would like to acknowledge Pfizer Inc. and Eli Lilly & Co. (through ACS Analytical Division) for their financial support during my graduate study. I was truly honored to be awarded both graduate fellowships. I am especially thankful to two special friends, Dr. John Earle and Mrs. Eleanor Earle. You have been family to us since Aimin and I came to this country. Thank you for all the love and care. Thank you for the time and encouragement you have given me in preparing this dissertation. Finally, I would like to acknowledge the support and encouragement of all my families and friends. iv TABLE OF CONTENTS DEDICATION.............................................................................................................. ii ACKNOWLEDGEMENTS ....................................................................................... iii LIST OF TABLES .................................................................................................... viii LIST OF FIGURES .................................................................................................... ix ABSTRACT................................................................................................................ xii CHAPTER 1. DNA SEQUENCING BY GEL ELECTROPHORESIS...................1 1.1. DNA Basics .........................................................................................................1 1.1.1. The Human Genome Project.........................................................................1 1.1.2. The Structure of DNA...................................................................................2 1.1.3. Shotgun Sequencing Strategies for Whole Genome Processing...................6 1.1.4. Sanger Chain Termination Sequencing for Determining the Primary Structure of DNA....................................................................................................8 1.2. DNA Separation by Gel Electrophoresis...........................................................11 1.2.1. Gel Electrophoresis Basics..........................................................................11 1.2.2. Electrophoresis Platforms for DNA Sequencing ........................................13 1.2.2.1. Slab Gel Electrophoresis (SGE)...........................................................13 1.2.2.2. Capillary Gel Electrophoresis (CGE) ..................................................15 1.2.2.3. Microchip Gel Electrophoresis (µ-CGE).............................................20 1.2.3. DNA Migration Models..............................................................................22 1.2.4. DNA Separation Matrices...........................................................................25 1.2.4.1. Cross-linked Polyacrylamide...............................................................26 1.2.4.2. Linear Polyacrylamide.........................................................................27 1.2.4.3. Other Polymers ....................................................................................28 1.3. Detection Methods for DNA Sequencing..........................................................29 1.3.1. Autoradiographic Detection........................................................................29 1.3.2. Fluorescence Detection...............................................................................30 1.3.3. Dye-primer/Dye-terminator Sequencing Chemistry...................................31 1.4. DNA Sequencing Instruments ...........................................................................33 1.4.1. Slab Gel Sequencer .....................................................................................33 1.4.2. Capillary-based Sequencing Instruments....................................................34 1.4.3. Microchip-based Sequencing Instruments..................................................37 CHAPTER 2. MULTIPLEXED FLUORESCENCE DETECTION FOR DNA SEQUENCING............................................................................................................42 2.1. Theory of Fluorescence .....................................................................................42 2.1.1. Quantum Yields ..........................................................................................44 2.1.2. Fluorescence Lifetime.................................................................................44 2.1.3. Steady-state and Time-resolved Fluorescence............................................45 2.2. Measurement of Fluorescence Lifetimes...........................................................47 v 2.2.1. Principles of Time-correlated Single-photon Counting (TCSPC) ..............48 2.2.2. Instrumentation of TCSPC..........................................................................50 2.2.2.1. Light Sources .......................................................................................51 2.2.2.2. Detectors ..............................................................................................53 2.2.2.3. Electronics............................................................................................56 2.2.3. Data Analysis..............................................................................................58 2.3. Fluorescent Dyes for DNA Labeling and Sequencing.......................................61 2.3.1. Visible Fluorescent Dyes ............................................................................62 2.3.2. Near-IR Fluorescent Dyes...........................................................................69 2.4. Fluorescence-based DNA Sequencing Strategies..............................................76 2.4.1. Color Discrimination Methods ...................................................................77 2.4.1.1. Single-color/Four-lane .........................................................................77
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