Setting Up And Application Of Magnetic Tweezers Combined With Single-molecule Fluorescence Microscopy

When we look into the history and philosophy of science,it is inevitable to find that the research interests in life science changed from clusters to single molecules.The traditional experiments based on hundreds or thousands of molecules only provide us with the average or statistical results while the single-molecule technique,on the other hand,provides more intrinsic results of biochemical reaction details.With the developments in single-molecule techniques,researchers nowadays have more powerful approaches to study life science.In this thesis,we firstly discuss several commonly used single-molecule techniques and then focus on a detailed discussion about a fluorescence microscope and a magnetic trapping instruments I have built as a graduate student in IOP.The topics include machine shopping,optical system tuning and programming.In the hope that these discussions here would be helpful for those who want to built single-molecule instrument for their own laboratory.In order to study the relation between conformation changes of a protein and its function,we also built an instrument that combines single-molecule microscope with magnetic tweezers.We used this instrument to study the dynamics of nucleosome disassembly.The results show a promising future of this instrument in single-molecule studies.The first part of this thesis discusses about the building up of magnetic tweezers and its application in the study of DNA unwinding by Pif1 protein.The second part discusses about the building up of single-molecule microscope and its application in the study of RecA-mediated homologous recombination.The third part discusses about the combining of magnetic tweezers and fluorescence microscope and its application in the study of nucleosome disassembly.