Analysis of proteins by in vitro mutagenesis

The generation and characterization of mutants is an essential component of any study on structure-- function relationships. Knowledge of the threedimensional structure of a protein, RNA species, or DNA regulatory element (e.g. a promoter) can provide clues to the way in which they function but proof that the correct mechanism has been elucidated requires the analysis of mutants that have amino acid or nucleotide changes at key residues. Classically, mutants are generated by treating the test organism with chemical or physical agents that modify DNA (mutagens). This method of mutagenesis has been extremely successful, as witnessed by the growth of molecular biology and functional genomics, but suffers from a number of disadvantages. First, any gene in the organism can be mutated and the frequency with which mutants occur in the gene of interest can be very low. This means that selection strategies have to be developed. Second, even when mutants with the desired phenotype are isolated, there is no guarantee that the mutation has occurred in the gene of interest. Third, prior to the development of gene-cloning and sequencing techniques, there was no way of knowing where in the gene the mutation had occurred and whether it arose by a single base change, an insertion of DNA, or a deletion. As techniques in molecular biology have developed, so that the isolation and study of a single gene is not just possible but routine, so mutagenesis has also been refined. Instead of crudely mutagenizing many cells or organisms and then analyzing many thousands or millions of offspring to isolate a desired mutant, it is now possible to change specifically any given base in a cloned DNA sequence. This technique is known as site-directed mutagenesis. It has become a basic tool of gene manipulation, for it simplifies DNA manipulations that in the past required a great deal of ingenuity and hard work, e.g. the creation or elimination of cleavage sites for restriction endonucleases. The importance of site-directed mutagenesis goes beyond gene structure--function relationships for the technique enables mutant proteins with novel properties of value to be created (protein engineering). Such mutant proteins may have only minor changes but it is not uncommon for entire domains to be deleted or new domains added. #GeneticsExamQuestionsSolutions #MolecularBiology #mutagens #aminoAcid #protein #RNA #Iherb #DNA #genomics #genecloning #sitedirectedMutagenesis #gene #phenotype #Cancer #inVitro #geneManipulation