Elsevier, Journal of Molecular Catalysis B: Enzymatic, (84), p. 40-47
DOI: 10.1016/j.molcatb.2012.04.018
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Methods to generate random mutant libraries in directed evolution are limited in functional diversity generation. The Sequence Saturation Mutagenesis (SeSaM) method was reported as a four step random mutagenesis method overcoming the limitations of epPCR based mutagenesis methods. SeSaM targets in contrast to epPCR each nucleotide “equally” avoiding mutagenic hot spots, achieving subsequent mutations in a codon (up to 37.1%), and allowing to adjust mutational biases through employed universal bases. In this manuscript, we report an advanced SeSaM method in which a protocol was developed and optimized for implementing the R (ribavirin) base in a SeSaM experiment. The R-based protocol was subsequently combined with the original P-base SeSaM protocol. Combining P- and R-base allows in SeSaM experiments to generate transversions at all four nucleotides of a given sequence with an unmatched chemical diversity. Following the later strategy, we developed a combined P- (at A & G positions) and R-base (at T & C positions) protocol, nearly doubled in comparison to the SeSaM-P [27] the number of mutations that are unobtainable by epPCR and removed the requirement of a single stranded template in the SeSaM method.