Sequence saturation mutagenesis

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.

Synonymous substitutions lead to amino acid preservation or conservative mutations with similar physico-chemical properties such as size and hydrophobicity are strongly prevalent.

[5] Another advancement of the method was achieved by introduction of degenerate bases instead of universal inosine and the use of optimized DNA polymerases, further increasing the ratio of introduced transversions.

[6] This modified SeSaM-TV+ method in addition allows for and favors the introduction of two consecutive nucleotide exchanges, broadening strongly the spectrum of amino acids that may be substituted.

PCR products of Step 1 are cleaved specifically at the phosphorothioate bonds, generating a pool of single-stranded DNA fragments of different lengths starting from the universal primer.

Comparison of the amino acid substitution pattern obtainable using standard epPCR methods (single nucleotide substitutions with transition bias) and the Sequence saturation mutagenesis method (introducing consecutive nucleotide substitutions with an increased ratio of transversions).
Experimental steps for generation of a non-biased random mutagenesis library using the sequence saturation mutagenesis method.