RECOMBINANT DNA TECHNIQUES: CLONING AND MANIPULATION OF DNA - A Guide to Methods in the Biomedical Sciences - page 52

constructing proteins with improved stability or with totally new proper-
ties. Antibodies with improved specificity and affinity can be generated
for therapeutic purposes using site directed mutatagenesis (see Chap-
ter 4). Several methods have been developed for mutating cloned genes.
The most commonly used are briefly described below.
Oligonucleotide-directed mutagenesis
This method allows for the alteration of a DNA sequence in a specific
way, and was invented by M. Smith. An oligonucleotide with a mutation of
interest is hybridized to the corresponding template sequence in plasmid
or viral DNA, and extended using DNA polymerase. If a circular DNA
template, such as a plasmid, or single stranded phage template such
as M13 is used, the second strand DNA will extend to the primer and
DNA ligase can be used to ligate the DNA product, producing a double
stranded product. The DNA is then used to transform competent E. coli
bacteria. The mismatch is then repaired in bacterial cells, which fixes
the mutation for selection using standard recombinant DNA techniques.
The function of the mutant gene can then be tested by expressing it
in the appropriate cells or in an organism.
A variation of this method uses “degenerate” oligonucleotides cov-
ering the region of interest (these are synthesized by adding small
amounts of incorrect nucleotide precursors during synthesis of the
oligonucleotide). The mixture of oligonucleotides is annealed to the tem-
plate, double stranded DNA is synthesized, and individual clones are
isolated and characterized.
PCR-directed mutagenesis
This is a variation of oligonucleotide-directed mutagenesis, in which
oligonucleotides containing mutations of interest are used as primers
in a polymerase chain reaction (see below for explanation of PCR).
The primers often contain restriction sites (either native to the gene or
introduced for cloning purposes). The resulting DNA is then cloned using
standard methods.
Linker scanning mutagenesis
This is a method for introducing a number of clustered mutations in a
short stretch of DNA. It is primarily used to identify transcriptional regula-
tory regions of a gene. Generally, linker scanning mutants are generated
that contain individual clusters over a larger region in order to rapidly de-
termine which region(s) are important in regulating a gene's expression.