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, phosphorothioate nucleotides are inserted in the gene sequence, cleaved and the resulting fragments elongated by universal or degenerate nucleotides. These nucleotides are then replaced by standard nucleotides, allowing for a broad distribution of nucleic acid mutations spread over the gene sequence with a preference to transversions and with a unique focus on consecutive point mutations, both difficult to generate by other mutagenesis techniques. The technique was developed by Professor Ulrich Schwaneberg at Jacobs University Bremen and RWTH Aachen University.

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