Sequence Saturation Mutagenesis

Sequence Saturation Mutagenesis (SeSaM) is a chemo-enzymatic random mutagenesis method applied for the directed evolution of proteins and enzymes. The technique was developed by Professor Ulrich Schwaneberg at Jacobs University Bremen and RWTH Aachen University. 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.

SeSaM has been developed in order to overcome several of the major limitations encountered when working with standard mutagenesis methods based on simple error-prone PCR (epPCR) techniques. These epPCR techniques rely on the use of polymerases and thus encounter limitations which mainly result from the circumstance that only single, but very rarely consecutive, nucleic acid substitutions are performed and that these substitutions occur usually at specific, favored positions only. In addition, transversions of nucleic acids are much less likely than transitions and require specifically designed polymerases with an altered bias. These characteristics of epPCR catalyzed nucleic acid exchanges together with the fact that the genetic code is degenerated decrease the resulting diversity on the amino acid level. Synonymous substitutions lead to amino acid preservation or conservative mutations with similar physico-chemical properties such as size and hydrophobicity are strongly prevalent. By non-specific introduction of universal bases at every position in the gene sequence, SeSaM overcomes the polymerase bias favoring transitory substitutions at specific positions but opens the complete gene sequence to a diverse array of amino acid exchanges.

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