Mutagenesis is the process of introducing changes into the DNA sequence. It is used in studies of gene, and the resulting protein, function. Mutations may be:
- Specific (site-directed mutagenesis), e.g. to mutate a given nucleotide, the altered nucleotide being present in the primer, resulting in a changed amino acid or introduction of a new restriction site. Knowledge of the sequence is needed;
- Random, e.g. to study structure: function relationships of a protein. The original and mutated proteins can be compared to find the function of parts of the protein. The location and type of mutation is not predetermined. Is possible with no knowledge of the sequence.
Random mutagenesis can make use of DNA shuffling or error prone polymerases. DNA shuffling: the DNA is digested randomly using DNaseI, denatured and used in PCR with no primers.
Error-prone Polymerase: This method relies on the fact that Taq polymerase has no proof-reading abilities. Other changes are the use of an error-prone buffer in the reaction which has increased magnesium ion concentration and the added manganese ions. Altering the Mn2+ concentration will change the number of mutations per kilobase. The concentration of dTTP is increased and dATP decreased.