Novel Method for Gene Function Analysis
BackgroundTraditional methods used to determine theinvolvement of a specific amino acid or group of amino acids to a protein’s structure,stability or function, are alanine scanning or random mutagenesis. However, these approaches are significantlylimited in accessing a residues contribution. With alanine scanning, the technique only measures the function of an aminoacid change to alanine, ignoring 18 other possible amino acids in the samecontext. Moreover, random mutagenesis, such as PCR and gene shuffling, arerecognized as being error-prone. TechnologyDr. Daniel Bolon’s laboratory at UMass Medical School has developed anovel method to analyze the function of all possible amino acid replacements atmultiple positions of a protein. Thismethod includes a platform to systematically generate and introduce randomcodons in a gene, mutational selection based on biochemical function orproperty, measure abundance of randomized sequence, and classify each codonreplacement. For enhanced accuracy, Dr.Bolon has also designed an accompanying software program for the automateddesign of oligonucleotides that can be used to mutagenize the DNA andfacilitate deep-sequencing readout and abundance of each mutation in thelibrary. Applications Enhancement of protein function, biological orchemical or properties. Identification of region(s) in a protein sensitiveor resistant to mutation(s).Determine which amino acid substitutions lead todrug or antibody resistance or sensitivity.Detection of amino acid(s) involved as active orreceptor binding sites.Competitive Advantages Provides improved coverage of the mutational spaceover existing methods by systematically substituting all possible amino acids ateach position(s) of interest. Cost-effective and greater efficiency- reducedexperimental time and labor- with maximum results.
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