Saturation 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.[1]

Method

Saturation mutagenesis is commonly achieved by artificial gene synthesis, with a mixture of nucleotides used at the codons to be randomised.[2] Different degenerate codons can be used to encode sets of amino acids.[1] 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).[1]

Degenerate codon No. of codons No. of amino acids No. of stops Amino acids encoded
NNN 64 20 3 All 20
NNK / NNS 32 20 1 All 20
NDT 12 12 0 RNDCGHILFSYV
DBK 18 12 0 ARCGILMFSTWV
NRT 8 8 0 RNDCGHSY

Applications

Saturation mutagenesis is commonly used to generate variants for directed evolution.[3][4]

See also

References

  1. 1 2 3 Reetz, M. T.; Carballeira J. D. (2007). "Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes". Nature Protocols. 2 (4): 891–903. PMID 17446890. doi:10.1038/nprot.2007.72.
  2. ↑ Reetz, Manfred T.; Prasad, Shreenath; Carballeira, José D.; Gumulya, Yosephine; Bocola, Marco (2010-07-07). "Iterative Saturation Mutagenesis Accelerates Laboratory Evolution of Enzyme Stereoselectivity: Rigorous Comparison with Traditional Methods". Journal of the American Chemical Society. 132 (26): 9144–9152. ISSN 0002-7863. PMID 20536132. doi:10.1021/ja1030479.
  3. ↑ Chica, Robert A.; et al. (2005). "Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design". Current Opinion in Biotechnology. 16 (4): 378–384. PMID 15994074. doi:10.1016/j.copbio.2005.06.004.
  4. ↑ Shivange, Amol V; Marienhagen, Jan; Mundhada, Hemanshu; Schenk, Alexander; Schwaneberg, Ulrich (2009-02-01). "Advances in generating functional diversity for directed protein evolution". Current Opinion in Chemical Biology. Biocatalysis and Biotransformation/Bioinorganic Chemistry. 13 (1): 19–25. doi:10.1016/j.cbpa.2009.01.019.
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