Site directed mutagenesis allows one to introduce virtually any desired mutation
ID: 53880 • Letter: S
Question
Site directed mutagenesis allows one to introduce virtually any desired mutation in a specific gene. One very useful application of the technique involves modifying a particular protein and evaluating the effect on biological or chemical activity. In the past, two methods have been used for producing modified proteins. One is to use chemical agents or ultraviolet light to induce mutations that result in changes in the amino acid sequence of proteins. The other is to modify certain residues in an isolated protein by treatment with chemical reagents; an example is the inactivation of a reactive serine in the active site of proteolytic enzymes like chymotrypsin, using diisopropylflurophosphate.
a) Why is site-specific mutagenesis superior to the two older procedures described above?
b) In order to carry out the modification of a protein using site-specific mutagenesis in the most efficient way, what sort of information should you have about the protein you wish to modify?
Explanation / Answer
a) Site-specific (SS) mutagenesis is superior to the older procedures because both of these procedures are only for specific set of nucleotides or amino acids. Ultraviolet light induces the formation of pyrimidine dimers including Thymine and Cytosine in a non-specific way. Similarly Diisopropylfluorophosphate inhibits serine proteases such as trypsin and chymotrypsin with no specificity with respect to the location within the protein. On the other hand SS mutagenesis can exclusively mutate the desired nucleotide at a given site which leads to the mutated amino acid incorporated in the protein. Thus, it is specific and highly efficient.
b) The mutation should be made in such a way that it does not affect the functioning of the protein due to stability or other issues. The effect on the protein should solely be because of the mutation so that the effect of the amino aicd can be studied. Hence the primary and secondary structure should be known whether the mutated amino acid doesn't collapse the protein on the whole. The sequence where the amino acid is mutated should be known properly whether there is any crucial sihnal or functional sequence. Most importantly the genome sequence should be known to mutate the protein accordingly.
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