Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2001 Analysis of near-infrared dye-labeled Sanger sequencing fragments with gel electrophoresis using the time-resolved flourescence lifetime indentification methods Suzanne Jeanel Lassiter 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 Lassiter, Suzanne Jeanel, "Analysis of near-infrared dye-labeled Sanger sequencing fragments with gel electrophoresis using the time- resolved flourescence lifetime indentification methods" (2001). LSU Doctoral Dissertations. 2957. https://digitalcommons.lsu.edu/gradschool_dissertations/2957 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]. ANALYSIS OF NEAR-INFRARED DYE-LABELED SANGER SEQUENCING FRAGMENTS WITH GEL ELECTROPHORESIS USING TIME-RESOLVED FLUORESCENCE LIFETIME IDENTIFICATION METHODS 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 Suzanne Jeanel Lassiter B.S., Georgia Southwestern State University, 1996 December, 2001 Acknowledgements Mommy and Dad: Thanks for all the restful weekends on the beach and the patience that you showed through all of the trying times. Your love and support helped get me were I am today. Jim: Without you I would not still have my sanity. I can’t tell you how much the IMs and e-mails got me through each day. I love you and can’t wait to enjoy the rest of our lives! Michelle: The best roommate a sister can have. Glad we got to make up for all the times we couldn’t get along when we were younger. Dr. Soper: Thank you for the encouragement and knowledge you bestowed upon me. I may have given up long ago if you had not been my advisor. Alyssa: Thanks for the funny stories and useful conversations. You can’t imagine how our bitch sessions helped me relieve the frustrations. Wieslaw: Without you I would still be going through my data point-by-point. Thank- you for all of the intellectual conversations and helping me solve problems. The “Super” Soper Group: I will remember you all. From the beginning to the end there was always one of you there to help lift my spirits when things got rough, especially Christie, Shawn, Shelby, Musundi, Michelle, Angela and also the adopted members and post-docs, Michele and Seok-Kwan. ii Table of Contents Acknowledgements .................................................................................................. ii Table of Contents....................................................................................................iii List of Tables ...................................................................................................................... vi List of Figures ........................................................................................................ vii Abstract ................................................................................................................... ix Chapter 1. DNA Sequencing Using Fluorescence Detection Methods................ 1 1.1. Genome Structure................................................................................... 1 1.2. Methods for Determining the Primary Structure of DNA ..................... 3 1.2.1. Maxam-Gilbert Sequencing........................................................... 6 1.2.2. Sanger Chain Termination Method ............................................... 9 1.3 Modes of Electrophoresis ...................................................................... 12 1.3.1. Slab Gel Electrophoresis ............................................................. 15 1.3.2. Capillary Electrophoresis............................................................. 18 1.4. DNA Migration..................................................................................... 23 1.4.1. Ogston Model .............................................................................. 23 1.4.2. Reptation Model .......................................................................... 24 1.4.3. Biased Reptation Model .............................................................. 24 1.5. Detection Methods for DNA Sequencing............................................. 25 1.5.1. Audioradiographic Methods........................................................ 25 1.5.2. Fluorescence Detection................................................................ 27 1.5.2.1. Fluorescent Dyes for DNA Labeling and Sequencing........... 33 1.5.2.2. Visible Fluorescent Dyes for DNA Labeling......................... 35 1.5.2.3. ET Dyes for DNA Sequencing .............................................. 39 1.6. Instrumental Formats for Fluorescence Detection in DNA Sequencing............................................................................. 43 1.6.1. Fluorescence Scanning Instruments ............................................ 46 1.6.2. Fluorescence Imaging Systems for DNA Sequencing................. 48 1.6.3. Fluorescence Detection Formats.................................................. 50 1.7. Sequencing Strategies........................................................................... 54 1.7.1. Single-color/four-lane.................................................................. 54 1.7.2. Single-color/single-lane............................................................... 56 1.7.3. Two-color/single-lane.................................................................. 57 1.7.4. Four-color/single-lane ................................................................. 60 1.8. References ............................................................................................ 62 iii Chapter 2. Time-Correlated Single Photon Counting ................................. 66 2.1 Fluorescence .......................................................................................... 66 2.2 Time-Resolved Spectroscopy................................................................ 68 2.3 TCSPC Instrumentation......................................................................... 72 2.3.1 Light Sources................................................................................ 72 2.3.1.1 Ti:sapphire Laser .................................................................... 75 2.3.1.2 Semiconductor Laser .............................................................. 78 2.3.2 Detectors....................................................................................... 82 2.3.2.1 Single Photon Avalanche Diodes ........................................... 82 2.4 TCSPC Electronics................................................................................ 85 2.4.1 Discriminators .............................................................................. 85 2.4.2 Time-to-Amplitude Converter (TAC) .......................................... 87 2.4.3 Multichannel Analyzer ................................................................. 87 2.5 Data Analysis......................................................................................... 88 2.5.1 Non-Linear Least Squares ............................................................ 88 2.5.2 Rapid Determination Method (RLD) ........................................... 89 2.5.3 Maximum Likelihood Estimator (MLE) ...................................... 89 2.6 Single-color/four-lifetime Sequencing .................................................. 91 2.7 Research Focus...................................................................................... 97 2.8 References ............................................................................................. 98 Chapter 3. Time-Resolved Fluorescence Imaging of Slab Gels for Lifetime Base-Calling in DNA Sequencing Applications.......................... 101 3.1. Introduction ........................................................................................ 101 3.2 Experimental Section........................................................................... 103 3.2.1 Instrumentation........................................................................... 103 3.2.2 DNA Sequencing........................................................................ 110 3.2.3 Data Analysis.............................................................................. 113 3.3 Results and Discussion........................................................................ 114 3.4 Conclusions ........................................................................................ 126 3.5 References ........................................................................................... 127 Chapter 4. Optimization of Sequencing Conditions for Near-IR Dye-Primers and Dye Terminators........................................................................... 130 4.1 Near-Infrared Dyes for DNA Sequencing ........................................... 130 4.2 Experimental Section........................................................................... 132 4.2.1 Capillary Preparation.................................................................. 132 4.2.2 Instrumentation..........................................................................