Saturated mutagenesis

Saturation mutagenesis is a random mutagenesis technique, in which a single codon or set of codons is randomised to produce all possible amino acids at the position. Saturation mutagenesis is a random mutagenesis technique, in which a single codon or set of codons is randomised to produce all possible amino acids at the position. Saturation mutagenesis is commonly achieved by artificial gene synthesis, with a mixture of nucleotides used at the codons to be randomised. Different degenerate codons can be used to encode sets of amino acids. Because some amino acids are encoded by more codons than others, the exact ratio of amino acids cannot be equal. Additionally, it is usual to use degenerate codons that minimise stop codons (which are generally not desired). Consequently, the fully randomised 'NNN' is not ideal, and alternative, more restricted degenerate codons are used. 'NNK' and 'NNS' have the benefit of encoding all 20 amino acids, but still encode a stop codon 3% of the time. Alternative codons such as 'NDT', 'DBK' avoid stop codons entirely, and encode a minimal set of amino acids that still encompass all the main biophysical types (anionic, cationic, aliphatic hydrophobic, aromatic hydrophobic, hydrophilic, small). In the case there is no restriction to use a single degenerate codon only, it is possible to reduce the bias considerably. Several computational tools were developed to allow high level of control over the degenerate codons and their corresponding amino acids. Saturation mutagenesis is commonly used to generate variants for directed evolution.