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Transversion‐enriched sequence saturation mutagenesis (SeSaM‐Tv + ): A random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error‐prone PCR
Author(s) -
Seng Wong Tuck,
Roccatano Danilo,
Loakes David,
Tee Kang Lan,
Schenk Alexander,
Hauer Bernhard,
Schwaneberg Ulrich
Publication year - 2008
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200700193
Subject(s) - transversion , saturated mutagenesis , directed evolution , mutagenesis , biology , nucleotide , genetics , taq polymerase , mutant , microbiology and biotechnology , transition (genetics) , polymerase , dna , polymerase chain reaction , mutation , gene , thermus aquaticus
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→T) and 6.38–9.69% (G→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 10 5 –10 6 ‐fold compared to epPCR. Finally, 16.7% of all sequenced mutants contained consecutive nucleotide exchanges composed mainly of a transversion followed by a transition.