Many methods exist by which mutations can be introduced into a specific region of DNA sequence. Also, techniques are available to introduce specific mutations into a site where a restriction endonuclease can act. In general, two methods are available to introduce mutations at specific sites. The first involves the use of the single-stranded bacteriophage Ml 3. From the gene sequence of the protein around the residue to be mutated a nucleotide sequence encompassing two or three amino acid residues either side of the altered codon is derived and the oligonucleotide chemically synthesized.
Some approaches employ two primers, but the basic theory is the same.
Oligonucleotide site-directed mutagenesis can also be accomplished using doublestranded plasmid DNA in a variety of ways. In all cases, the gene to be mutated is cloned into a plasmid vector. The same approach is then taken as with the single-stranded phage mutagenesis. Klenow, dNTPs, ATP, and ligase are added and the resultant plasmid is introduced into an appropriate E. coli host. The mismatch in the plasmid is handled in a similar manner as that in the single-stranded phage.