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Site saturation mutagenesis (SSM), or simply site saturation, is a random mutagenesis technique used in protein engineering, in which a single codon or set of codons is substituted with all possible
Site saturation mutagenesis (SSM), or simply site saturation, is a random mutagenesis technique used in protein engineering, in which a single codon 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 a ...
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 bysite-directed mutagenesis
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...
PCR with a randomised codon in the primers (e.g. SeSaM Sesam, SESAM or SeSaM may refer to:
* SESAM (database), a relational database developed by Fujitsu Siemens
* SESAM (FEM), a structural analysis software
* Sesam (search engine), a Scandinavian internet search engine
* SeSaM-Biotech GmbH, a biotech ...
) or by artificial gene synthesis
Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides '' de novo''. Unlike DNA synthesis in living cells, artificial gene synthesis d ...
, 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 biomolecule ...
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 fordirected evolution
Directed evolution (DE) is a method used in protein engineering that mimics the process of natural selection to steer proteins or nucleic acids toward a user-defined goal. It consists of subjecting a gene to iterative rounds of mutagenesis ( ...
.
See also
*Sequence Saturation Mutagenesis Sequence saturation mutagenesis (SeSaM) is a chemo-enzymatic random mutagenesis method applied for the directed evolution of proteins and enzymes. It is one of the most common saturation mutagenesis techniques. In four PCR-based reaction steps, p ...
References
{{DEFAULTSORT:Saturated Mutagenesis Genetics techniques Molecular genetics Mutagenesis