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= Deep Mutational Scanning =

Deep Mutational Scanning couples selective pressures on protein variants with High-Throughput Sequencing (HGS) in order to measure the functional levels of the variant proteins. This technique can be utilized for the selection of substrate binding affinity, binding of peptides, stability, and catalysis. High-Throughput Sequencing works by measuring the frequency of the protein sequence as a measure of the protein’s level of function. '''Frequency is used as a measure of function because the organisms with the more efficient protein would grow more than its less efficient counterpart and therefore have more copies of the better protein. When measuring the variant protein for changes solubility and stability it is also possible to attach a fluorescent protein to the target protein and measure the intensity of fluorescence.''' This method works by changing the DNA that codes for a single amino acid in a protein to create a sequence for every different amino acid at a specific spot. This process creates different forms of the proteins with slight variations at a single location. All the variant sequences are placed in a plasmid or virus and cloned into a target organism. Then HGS is used on the cloned organisms prior to selection in order to gain a baseline of function. Following this a selective pressure for the specific protein is used and HGS is used again to compare to the pre-selection frequencies. Variants with higher changes in frequencies have higher levels of functionality compared to proteins with lower frequencies. '''This method is used to build a library based on a single change in an amino acid, but it is possible to combine multiple changes from different locations of high frequency can create a more stable and soluble protein. However, this is true for amino acid sequence away from the catalytic core of the protein.'''