Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR.
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(+)). dNTP alpha S 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 transversion-enriched protocol has been developed. In a mutant library generated using dGTP alpha S 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.
Wong
Tuck Seng
TS
Jacobs University Bremen, School of Engineering and Science, Bremen, Germany.
Roccatano
Danilo
D
Loakes
David
D
Tee
Kang Lan
KL
Schenk
Alexander
A
Hauer
Bernhard
B
Schwaneberg
Ulrich
U
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics*
D011994:Recombinant Proteins; D004247:DNA