DNA Tether Assays and Flow Testing: Increasing Reproducibility and Throughput in Single Molecule Experiments Open Access

Martin, Stefano (Spring 2021)

Permanent URL: https://etd.library.emory.edu/concern/etds/7h149r25w?locale=en
Published

Abstract

DNA transcription is essential to most, if not all cellular life. Transcription and gene expression are regulated by proteins called transcription factors, and DNA torsion and tension affect the binding and unbinding of transcription factors. Single molecule experiments allow us to study the mechanics of single molecules of DNA, something that is not possible under experimental conditions that hope to extract data from Avogadro’s number scales. Techniques like tethered particle microscopy (TPM) and magnetic tweezers (MT) allow us to perform single molecule experiments and collect data, relying on DNA tether assays to perform experimentation. DNA tether assays involve anchoring a linear segment of DNA to an observation chamber and attaching the other end to a microscopic bead that can be manipulated and tracked. Some single molecule experiments, like the ones planned for the future in the Finzi lab, require multiple instances of buffer exchange within the chamber between data collection steps. This thesis shows a typical Magnetic Tweezer experiment and the limitations of the employed methods for tether assay assembly, manipulation and fluid exchange. This thesis also describes the integration of a pump-assisted flushing system and development of corresponding software to extract data stored in video formats. The ability of a construct of DNA to stretch, twist, and be inducible to supercoiling was tested and the construct was used in further testing with the aim of mitigating the adverse effects of uncontrolled and non-reproducible fluid exchange rates within the observation chambers. 

Table of Contents

Table of Contents:

Chapter I: Introduction

A. The Central Dogma and DNA (pg. 1)

B. DNA Topology and Supercoiling (pg. 4)

C. Single Molecule Experiments and DNA Tether Assays (pg. 8)

D. Experimental Goals and Significance (pg. 9)

Chapter II: Experimental Planning

A. Tether Preparation (pg. 10)

B. Flow Chamber Preparation (pg. 17)

C. Tethered Particle Tracking and Magnetic Tweezers (pg. 19)

D. Flow Tests: Bulk Flow & Bead Displacement Tracking (pg. 23)

Chapter III: Results

A. Magnetic Tweezer Tests (pg. 26)

B. Flow Tests using Tissues and Micropipettes (pg. 28)

C. Flow Tests with Pump Implementation (pg. 30)

Chapter IV: Discussion

A. Making A New DNA Tether for MT Measurements of Protein-Constrained Torsion (pg. 35)

B. Implementation of a Pump Flow System for Reproducible Chamber Preparations (pg. 35)

C. Future Objectives and Outlook (pg. 38)

Chapter V: References (pg. 40)

Chapter VI: Appendix

A. pDD_1N3BbvCI Plasmid and Primer Sequences (pg. 43)

B. Video Player MATLAB Code (pg. 45)

C. Flow Chamber Assembly Protocols (pg. 48)

D. Calibration Slide and Pixel Conversion Factor (pg. 50)

Table of Figures:

Figure 1. Chemical Structures of Nucleic Acid Monomers and Base Pairing (pg. 3)

Figure 2. DNA Structure and Central Dogma (pg. 4)

Figure 3. Histone-DNA Complex and Lac Repressor (pg. 5)

Figure 4. Plectonemic DNA Supercoiling (pg. 7)

Figure 5. Polymerase Chain Reaction (pg. 11)

Figure 6. Restriction Endonuclease and Ligation Reaction (pg. 12)

Figure 7. Agarose Gel Electrophoresis (pg. 14)

Figure 8. pDD_1N3BbvCI Plasmid and DNA Tether Fragment PCR Results (pg. 15)

Figure 9. Fragment Ligation and Gel Confirmation (pg. 16)

Figure 10. Model Cross-Section of a Flow Chamber (pg. 18)

Figure 11. Diagram of B Induced Forces on Beads (pg. 20)

Figure 12. Magnetic Tweezer Microscope (pg. 22)

Figure 13. Leica Light Microscope (LM) and Pump System (pg. 23)

Figure 14. Custom Video Player GUI (pg. 25)

Figure 15. Magnetic Tweezing Experiments and Sample Field of View (pg. 27)

Figure 16. Micropipette-Tissue Sample Loading Strategy (pg. 29)

Figure 17. Flow Chamber and Slide Adapter Design (pg. 32)

Figure 18. Bead Average Velocities vs Buffer Exchange Strategy (pg. 34)

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