Gene synthesis by assembly of short oligonucleotides

ii Table of Contents. iii List of Tables . vi List of Figures . vii List of Abbreviations . viii Acknowledgements. ix Preface. x Dedication . xi 1 Introduction. 1 1.1 A DNA Primer . 1 1.2 Gene Synthesis. 2 1.2.1 Background. 3 1.2.2 Phosphoramidite Chemistry and Oligonucleotide Synthesis. 5 1.2.3 PCA and LCR . 8 1.2.4 Reducing Errors . 11 1.3 Rationale . 11 1.3.1 Ligase Requirements. 13 1.3.2 Library Size. 16 2 Characterization of T4 DNA Ligase and Short Oligonucleotides . 17 2.1 Methodology for Fluorescence Experiments . 17 2.1.1 Methodology for Bead Immobilization . 18 2.1.2 Methodology for Basic Ligation Assays . 19 2.1.3 Methodology for Ligation Assays With a Supplementary, Second Oligonucleotide. 20 2.1. 4 Methodology for Hexamer Ligation Assays with Serial Concentrations of PEG . 20 2.1. 5 Methodology for Characterizing Rescue Assays with Serial Concentrations of the Supplementary Oligonucleotide . 20 2.1. 6 Methodology for Ligation Assays with Alternative Rescue Oligonucleotides . 21 2.2 Fluorescence Experiments . 21 2.2.1 Minimal Oligonucleotide Lengths for Ligation. 21 2.2.2 Improvement of 4 bp Ligations with a second oligonucleotide. 23 2.2.3 Improvement of 3 bp Ligations with a Second Oligonucleotide . 24 2.2.4 Alterations in Oligonucleotide Arrangements Improve Ligation Efficiency 27 2.2.5 Optimal Supplementary Oligonucleotide Concentrations are Dependent on the Ligation Kinetics of the First Oligonucleotide . 30 2.2.6 Improved Ligation Rates in the Presence of PEG . 31 2.2. 7 Hairpin Oligonucleotides can Improve Hexamer Ligation. 32 2.3 Methodology for Pooled Oligonucleotide Experiments . 34 2.3.1 Methodology for Octamer and Hexamers Pools. 34 2.4 Pooled Oligonucleotide Experiments . 34 2.4.1 Octamers . 35 iv 2.4.2 Hexamers . 36 2.4.3 Dephosphorylated Oligonucleotides can be Used in Pooled Assemblies. 37 2.5 Methodology for DNA Construction Experiments. 39 2.5. 1 Experimental Methodology for Pair-wise Assembly. 39 2.5. 2 Experimental Methodology for Serial Assembly . 41 2.6 DNA Construction Experiments . 42 2.6. 1 Hierarchical Assembly of a 128 bp Construct . 43 2.6. 2 Serial Assembly of a 128 bp construct . 47 3 Algorithmic Assembly of Genes. 49 3.1 Sequence Tiling . 50 3.2 Problematic Regions . 50 3.2.1 Restriction Sites . 50 3.2.2 Complete Palindromes. 52 3.2.3 Repeats in Oligonucleotide Pools . 54 3.2.4 Palindromic 4 bp Ends . 56 3.2.5 Single Polynucleotide Stretches. 57 3.3 Conflict Avoidance Strategies . 58 3.3.1 Restriction sites. 59 3.3.2 Palindrome Avoidance. 60 3.4 Methodology of Algorithmic Assembly Strategies . 62 3.4.1 Subunit Requirement Strategies. 62 3.4.2 Subunit Assembly Strategies . 64 3.4.3 Solution Selection and Filtering Strategies. 66 3.5 Algorithmic Assembly of Selected Genes . 68 3.5. 1 Overview of the assembly process. 68 3.5. 2 Assembly of EGFP . 70 3.5. 3 Assembly of TetC . 74 4 Discussion . 76 4.1 Addressing Error Rates . 77 4.1.1 Error Rate Analysis. 78 4.1.2 Cost Analysis . 81 4.1.3 Reducing Error Rates. 84 4.1. 4 Assembly with Hexamer Analogues. 87 4.1. 5 Danger and Security. 88 4.1. 6 Conclusions. 89 4.2 Future Research . 89 4.2.1 Engineering a New Ligase through Directed Evolution . 89 4.2.2 Microfluidic and Labcyte Assembly. 90 4.3 Significance. 92 5 References. 93 Appendix A – Glossary. 96 Appendix B – Sequencing Results for Beta-Actin Assemblies . 99 B.1 Serial Assembly of 100bp BA. 100 B.1 Serial Assembly of 128bp BA using High Concentration Oligonucleotides . 102 B.3 Hierarchical (pair-wise) Assembly of 128bp BA. 103 Appendix C – Source Code fo