Saturation mutagenesis

Saturation mutagenesis is commonly achieved by site-directed mutagenesis PCR with a randomised codon in the primers (e.g. SeSaM)[2] or by artificial gene synthesis, with a mixture of synthesis nucleotides used at the codons to be randomised.

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).

[1] In the case there is no restriction to use a single degenerate codon only, it is possible to reduce the bias considerably.

[4][5] Several computational tools were developed to allow high level of control over the degenerate codons and their corresponding amino acids.

[6][7][8] Saturation mutagenesis is commonly used to generate variants for directed evolution.

Saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants at position 5 are shown below.
Depiction of one common way to clone a site-directed mutagenesis library (i.e., using degenerate oligos). The gene of interest is PCRed with oligos that contain a region that is perfectly complementary to the template (blue), and one that differs from the template by one or more nucleotides (red). Many such primers containing degeneracy in the non-complementary region are pooled into the same PCR, resulting in many different PCR products with different mutations in that region (individual mutants shown with different colors below).