dRTP and dPTP a complementary nucleotide couple for the Sequence Saturation Mutagenesis (SeSaM) method

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.