Saturation Mutagenesis
Also known as: complete mutagenesis, exhaustive mutagenesis
Creating every possible single amino acid substitution at every position in a protein — the prerequisite for deep mutational scanning.
Source: Wrenbeck EE et al. 'Deep sequencing methods for protein engineering and design.' Curr Opin Struct Biol 2017;45:36-44. https://doi.org/10.1016/j.sbi.2016.11.001
Primary reference ↗Saturation mutagenesis generates every possible single amino acid variant of a protein. For a protein of length L, this produces up to L × 19 variants (19 non-wild-type amino acids per position).
Scale Example
For CYP2C9 (490 amino acids):
- 490 positions × 19 substitutions = 9,310 possible single-point variants
- Plus synonymous codons, stop codons, and deletions in DNA-level libraries
- Amorosi 2021 measured 6,142 missense variants with reliable activity scores
Methods
Modern saturation mutagenesis uses:
- Oligo-directed recombination (e.g., MAGE) or error-prone PCR for library construction
- DNA synthesis with defined codon substitutions for precise control
- Deep sequencing (next-gen sequencing, NGS) to count variant frequencies
Relationship to DMS
Saturation mutagenesis creates the library; deep mutational scanning measures the library. DMS requires saturation mutagenesis as its first step.