Site-Directed Mutagenesis by Polymerase Chain Reaction

Site-directed mutagenesis (SDM) is a powerful tool for analyze protein structure and function, protein folding, and enzyme mechanism (1 ). Several protocols for SDM by polymerase chain reaction (PCR) are published (2 –8 ). Here, two protocols, which turned out to be robust and efficient in the authors’ hands, are described in detail. Both methods comprise two steps: In the first step the desired mutation is introduced by a PCR primer used to amplify one part of the target gene. In a second step, this PCR product is then used as a megaprimer to amplify the full gene containing the mutation. Both methods deviate in how the mutated gene is introduced back into its cloning vector: In method 1, the whole vector plasmid is amplified in a PCR reaction, method 2 relies on restriction enzyme cleavage of the gene and vector, followed by ligation. In both methods, together with the mutation, a restriction enzyme marker site is introduced into the target gene by silent mutations, to allow fast and convenient screening for the presence of the mutation (4 ,7 ,9 ). Because random mutagenesis and directed evolution are being widely used for protein engineering (10 ), a method for random mutagenesis by PCR is described, using spiked oligonucleotides, which contain a mixture of all four nucleotides, to a certain degree (11 ). This technique allows randomization of a small part of a gene, at a level that can be chosen at will, and is a convenient alternative to cassette mutagenesis methods described previously (12 ,13 ). The protocol described here allows the construction of large mutant libraries (10⁴ -10⁵ clones). Because wild-type (WT) alleles are efficiently excluded from transformants, screening for the presence of the mutational primer is not necessary.