Preparation of DNA-Structures in Single Molecule Experiments

Abstract

The study of biomolecules on a single molecule level enables the investigations of dynamic, mechanistic as well as heterogeneous behavior of the biomolecules. For studying the features of DNA-protein interactionsinvolved in DNA-repair, on the single-molecule level, the combination of microfluidics, surface preparations and fluorescence microscopyis usedby the single molecule biophysics group at the physics department at the University of Gothenburg. In addition, traditional biochemical techniques, Polymerase Chain Reaction and agarose-gel electrophoresis, are also used to produceand analyze various types of DNAused in the single molecule experiments. The workis interdisciplinary and combines physics, surface science, biochemistry and micro/nano science, which motivatedme to finish my master thesis project in the field of single molecule biophysics. In order to investigatethe repairofthe double-stranded break (DSB), we tried to simulate the homologous recombination (HR) process in vitro to study the interaction between DNA and the protein Rad51 through single molecule experiments. The objective of thisthesis project was to prepare DNA structures to mimic the damaged DNA with processed DSBs. The damaged and processed DNA structuresare double-stranded DNA (dsDNA) with a 3’-single-stranded DNA(ssDNA)overhang ofvariouslengths. These DNA structureswillwork as the substrates for the study of the DNA-protein interaction that are involved in the repairof DSBs. Short pieces of ssDNA (140nt) were designed and bought directly. Long pieces of ssDNA (5kb) were produced via Polymerase Chain Reaction (PCR) followedbylambda-exonuclease treatment using lambda-phage DNA(48 kbp)as template. The optimal conditions were acquired after repeated experiments and the results were verified by agarose gel electrophoresis. Finally, these ssDNA molecules were ligated to the originallambda-DNA. Fluorescently labeled Replication Protein A (RPA) that preferentially binds ssDNA, was used to confirm that the DNA samples contained the desired structure, dsDNA with 3’-ssDNA overhang. The interaction betweenDNA and RPA was studied inmicrofluidicflowcellsusingfluorescence microscopy where the DNA sampleswereattached to a functionalized lipid bilayer surfacewithin the microfluidic flowcells.