Biomedical Engineering Reference
In-Depth Information
4.2 million from the mouse, and tens to hundreds of thousands of se-
quences from a variety of other organisms. When an investigator iden-
tifies a partial sequence from a gene of interest, s/he can screen that
sequence against the EST database and obtain additional sequence
information for the same or related genes in a single organism, or in
different organisms. The EST database has been an invaluable tool to
identify the coding sequences in genes that span large amounts of ge-
nomic DNA, discover new members of gene families, and identify related
genes across species.
D.
Site Directed Mutagenesis
Introduction
Specific mutations in the DNA sequence of genes can result in the loss
of function of the encoded protein. The classical way of defining gene
function is to select for phenotypic changes, and then correlate these
with specific mutations. However, this is cumbersome and slow, since the
rate of mutation in genes is low (1 in base pairs per generation) and
many changes do not result in perceptible phenotypic changes in the
corresponding protein. A more rapid and productive method of introduc-
ing changes in genes is to experimentally mutate the gene, and then test
for changes in function that result from these mutations. This method,
called site directed mutagenesis, was the brainchild of Michael Smith,
who shared the Nobel Prize with K. Mullis in 1993 for this landmark
idea (22). Using site directed mutagenesis, one or more nucleotides can
be altered in a gene, resulting in changes in the corresponding amino
acid sequence of the encoded protein. Today, site directed mutagenesis
is used routinely to identify and characterize important regulatory ele-
ments of genes and to identify individual domains and specific amino
acids within protein domains that are responsible for their function. The
rapid advancement in our understanding of how enzymes function, the
identification of the templates recognized by enzymes, an understand-
ing of how proteins interact with each other or with their cognate DNA
sequences (in the case of transcription factors), the determination of
motifs in proteins that are crucial for protein folding and their three-
dimensional structures, and the “quality control” mechanisms within a
cell that govern protein folding and retention in the secretory pathway.
All of these advances can be traced to a large extent to the invention
and use of site directed mutagenesis.
Site directed mutagenesis is one of the most powerful methods
available for the analysis of protein structure:function relationships us-
ing recombinant DNA technology. It also has advanced possibilities of
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