Site-directed Mutagenesis

Site-directed mutagenesis has been widely used in the study of protein functions.  There are many approaches.  An oligonucleotide-based method is illustrated in Figure 9-G-1. This method was first developed by Michael Smith who was awarded a Nobel Prize in 1993 for this contribution. Site-directed mutagenesis can also be achieved by using PCR (more info).

Figure 9-G-1.  Illustration of the basic steps in a site-directed mutagenesis method.
(1) Cloning the DNA of interest into a plasmid vector.
(2) The plasmid DNA is denatured to produce single strands.
(3) A synthetic oligonucleotide with desired mutation (point mutation, deletion, or insertion) is annealed to the target region.  In this figure, the T to G point mutation is used as an example.
(4) Extending the mutant oligonucleotide using a plasmid DNA strand as the template.
(5) The heteroduplex is propagated by transformation in E. coli.

After propagation, in theory, about 50% of the produced heteroduplexes will be mutants and the other 50% will be the "wild type" (no mutation).  In commercial mutagenesis kits, some selection and enrichment methods have been used to favor the production of mutants.

Book section

In vitro site-specific mutagenesis - From Human Molecular Genetics, 1999.