Site saturation mutagenesis (SSM), or simply site saturation, is a

random mutagenesis In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various ...

technique used in

protein engineering Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to im ...

, in which a single

codon The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...

or set of codons is substituted with all possible

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...

s at the position. There are many variants of the site saturation technique, from paired site saturation (saturating two positions in every mutant in the library) to scanning site saturation (performing a site saturation at every site in the protein, resulting in a library of size 0 x (number of residues in the protein)that contains every possible point mutant of the protein).

Method

Saturation mutagenesis is commonly achieved by site-directed mutagenesis PCR with a randomised codon in the primers (e.g. SeSaM) or by artificial gene synthesis, with a mixture of synthesis

nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecul ...

s 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.

Applications

Saturation mutagenesis is commonly used to generate variants for directed evolution.

References

{{DEFAULTSORT:Saturated Mutagenesis Genetics techniques Molecular genetics Mutagenesis

Method

Applications

See also

References