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Restriction endonucleases are enzymes that cleave double stranded DNA and are widely used in recombinant DNA technology, yet much about their mechanisms of action is poorly understood. It is the goal of this research to fill the information gap on restriction endonucleases and increase our understanding of how these enzymes work by addressing the lack of kinetic data on type II restriction ... read moreendonucleases. A novel fluorescence-based approach was used to track the progress of the cleavage of short synthetic DNA oligonucleotides by restriction endonucleases in real time. This approach was used to determine kinetic parameters of EcoRV and PvuII in bulk solution and adapted to a fiber-optic array platform that would allow for single-molecule kinetic data. Ultimately this research will lead to single-molecule observations of restriction endonuclease activity, which will give greater insight into their mechanisms than bulk data can provide. A fiber-optic based platform was also developed to screen restriction endonucleases for aberrant activity that is often observed under nonstandard reaction conditions.read less
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