title: SeSaM-Tv-II generates a protein sequence space that is unobtainable by epPCR.
abstract: Generating high-quality mutant libraries in which each amino acid is equally targeted and substituted in a chemically diverse manner is crucial to obtain improved variants in small mutant libraries. The sequence saturation mutagenesis method (SeSaM-Tv(+) ) offers the opportunity to generate such high-quality mutant libraries by introducing consecutive mutations and by enriching transversions. In this study, automated gel electrophoresis, real-time quantitative PCR, and a phosphorimager quantification system were developed and employed to optimize each step of previously reported SeSaM-Tv(+) method. Advancements of the SeSaM-Tv(+) protocol and the use of a novel DNA polymerase quadrupled the number of transversions, by doubling the fraction of consecutive mutations (from 16.7 to 37.1 %). About 33 % of all amino acid substitutions observed in a model library are rarely introduced by epPCR methods, and around 10 % of all clones carried amino acid substitutions that are unobtainable by epPCR.
authors.affiliation: Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Hemanshu
authors.initials: H
authors.lastname: Mundhada
authors.forename: Jan
authors.initials: J
authors.lastname: Marienhagen
authors.forename: Andreea
authors.initials: A
authors.lastname: Scacioc
authors.forename: Alexander
authors.initials: A
authors.lastname: Schenk
authors.forename: Danilo
authors.initials: D
authors.lastname: Roccatano
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D015723:Gene Library; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*
chemical_list: D011506:Proteins