Site-Directed Mutagenesis Demonstrates Possibilities for Restriction Site Mutations
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The process of site-directed mutagenesis allows us to target specific amino acid sequences in plasmid DNA in vitro. Previous mutagenic techniques did not allow for specific mutations to take place, but site-directed mutagenesis, which uses plasmid vectors, has dramatically improved our ability to manipulate DNA. Several forms of site-directed mutagenesis have been developed since the years of its conception, each technique specific to certain conditions. Using these new techniques, were are now able to direct at which amino acid sequence we desire mutations to occur, greatly increasing our understanding of many genetic mechanisms which were previously not understood. Previous research has shown that the 131 st amino acid residue of this sequence, normally a cysteine, plays a critical role in the biological activity of the hrFGF-l gene. A previous mutation of this cysteine to a serine dramatically increased the activity of the gene, proving the importance of this position in the hrFGF-l gene. The DNA is mutated through the unique restriction site elimination technique proposed by Deng and Nickoloff, and is then transformed into mismatch repair defective E. coli bacteria. Transformants are screened through a restriction digest of the unique, nonessential restriction site, and are then retransformed into an appropriate host. Linearized parental molecules transform bacteria inefficiently, and the desired mutation can be recovered at frequencies approaching 80% (Deng & Nickoloff, 1992). The procedure used employs simple protocols, uses inexpensive materials, and can be performed in as little as 48 hours.