Date of Award
Master of Science (MS)
In this study, a detailed description of the optical tweezers microscopy technique is presented, as well as the methodologies used to prepare the DNA molecule for mechanical measurements at the nanoscale. The main objective is to initiate and extend the experimental biophysical studies on the DNA-proteins interactions at the University of Texas at Brownsville (UTB). DNA-binding proteins control almost all aspects of cellular function, such as transcription; chromosome maintenance, replication and DNA repair depend on the interaction of proteins with DNA. In view of such an important role played by DNA–protein interactions, various techniques have evolved over the years to elucidate them. Each technique, with its own advantages and drawbacks, serves a very specific purpose. The optical tweezers has evolved as one of the powerful tools for studying the DNA–Protein complexes at a single molecule level. It allows to characterize the mechanisms involved in DNA–protein complex formation in different conditions with high resolution. It quantitatively identifies protein position along DNA molecules, DNA flexibility, curvature and conformational change after protein binding. This thesis describes the design and calibration of the optical tweezers. We measure relative displacements with nanometer accuracy and forces with an accuracy of 10%. The capability of the instrument is demonstrated by stretching a single molecule of DNA because of the elasticity of DNA has previously been well characterized. Every DNA sample used in this study has been engineered biochemically in order to accomplish proper linkage between the biological system and its supports.
Breaking down the main problem leads us to four different aspects, optical tweezers, engineered molecules, coupling molecules/supports system, and the gathering of data. There are a variety of methods used to approach these problems. For the optical tweezers we will be mainly dealing with the calibration of objectives, lasers, stage control, trapping a bead and tracking the bead. Sample preparation involves polymerase chain reaction (PCR), spectrophotometer analysis, DNA electrophoresis, DNA purification process, DNA binding tests, Dot Blot Analysis, measuring of size of particles, zeta potentials, and multimode reader. We are able to confirm visually through the microscope a complete bond system by engaging all results from the experiments.
We consider that our study will open up new and exciting research opportunities at UTB to study biological interactions at the level of single molecules. Also our system will be a very useful equipment to demonstrate to local students the physical principals of optics applied to biological systems.
University of Texas Brownsville