Site-Directed Mutagenesis Demonstrates Possibilities for Restriction Site Mutations
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Authors
Krohn, Nathan
Issue Date
1998
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
i. 14 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.