SEARCH

SEARCH BY CITATION

Keywords:

  • Directed evolution;
  • Mutagenesis Assistant Program;
  • Random mutagenesis;
  • Sequence saturation mutagenesis;
  • Transversion

Abstract

The sequence saturation mutagenesis (SeSaM) method has been advanced to a random mutagenesis method with adjustable mutational biases. SeSaM offers, for example, a bias that is complementary to error-prone (ep) PCR and is enriched in transversions (SeSaM-Tv+). dNTPαS and three degenerate bases (P, K and I) are used to control mutational bias flexibly. After quantifying incorporation rates of dPTP, dKTP and dITP by terminal transferase using a luciferase-based assay and investigating the read and/or write activities of eight DNA polymerases, a transversionenriched protocol has been developed. In a mutant library generated using dGTPαS and dPTP, transversion frequencies of 16.22–22.58% (G[RIGHTWARDS ARROW]T) and 6.38–9.69% (G[RIGHTWARDS ARROW]C) were achieved. These mutational spectra are complementary and occur twice as frequently in comparison to standard epPCR methods employing Taq DNA polymerase. For generating more complex mutant libraries, the occurrence of consecutive nucleotide exchanges was increased by 105–106-fold compared to epPCR. Finally, 16.7% of all sequenced mutants contained consecutive nucleotide exchanges composed mainly of a transversion followed by a transition.