Developing New Substrates for a Single-Molecule DNA Cleavage Assay
English (United States)
In this experiment, the overall goal is to measure Fo¨rster Resonance Energy Transfer (FRET) efficiency between individual restriction endonuclease molecules and deoxyribonucleic acids (DNA) with the aid of Total Internal Reflection Fluorescence (TIRF) microscopy. Fo¨rster Resonance Energy Transfer (FRET) is a phenomenon in which energy is transferred between two fluorescent markers through dipole-dipole coupling, without the emission of a photon by the donor. Measurements of FRET efficiency are an excellent method to determine distances between molecules because energy transfer is extremely sensitive to small changes in distances between the donor and the acceptor. A fluorescent marker placed on a desired protein will serve as the acceptor, while a quantum dot (Q-dot) coupled to DNA will function as the donor. When excited with light, the quantum dot can transfer energy, which subsequently excites the acceptor. Quantum dots, which are semiconductor nanocrystals, are used in these experiments because their high degree of photostability permits extended observation times not possible with traditional fluorescent molecules. In order to successfully observe DNA molecules using TIRF microscopy and determine FRET efficiency, a quantum dot needs to be coupled properly to a DNA molecule that can be captured on a coverslip for observation. Quantum dots are coupled to the DNA using a cross-linker molecule that reacts with a sulfhydryl group on the DNA and a primary amine on the quantum dot. The quantum dot-labeled DNA is then annealed to a strand of biotinylated DNA. Finally, the completed duplex DNA construct is flowed through the channel of a microfluidic flow cell constructed on a chemically functionalized coverslip, and image analysis is used to probe the specificity of binding between the biotinylated DNA and the coverslip. Using this approach, conditions for cross-linking can be optimized to aid in generating a variety of DNA constructs that can be used to characterize protein-DNA interactions.
Mendez, Luz Caroline, "Developing New Substrates for a Single-Molecule DNA Cleavage Assay" (2017). Honors Theses - All. 1901.
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