title: Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.
abstract: Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries.
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland.
authors.forename: Soili I
authors.initials: SI
authors.lastname: Lehtonen
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Fimlab Laboratories, Biokatu 4, FI-33520 Tampere, Finland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Taskinen
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Next Biomed Technologies NBT Oy, Viikinkaari 6, FI-00790 Helsinki, Finland.
authors.forename: Elina
authors.initials: E
authors.lastname: Ojala
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Fimlab Laboratories, Biokatu 4, FI-33520 Tampere, Finland.
authors.forename: Sampo
authors.initials: S
authors.lastname: Kukkurainen
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Fimlab Laboratories, Biokatu 4, FI-33520 Tampere, Finland.
authors.forename: Rolle
authors.initials: R
authors.lastname: Rahikainen
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland.
authors.forename: Tiina A
authors.initials: TA
authors.lastname: Riihimäki
authors.affiliation: The Center for Infectious Medicine, Department of Medicine HS, Karolinska Institute, Karolinska University Hospital, Huddinge F59, SE-141 86 Stockholm, Sweden.
authors.forename: Olli H
authors.initials: OH
authors.lastname: Laitinen
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Tampere University Hospital, FI-33521 Tampere, Finland.
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.affiliation: BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Fimlab Laboratories, Biokatu 4, FI-33520 Tampere, Finland vesa.hytonen@uta.fi.
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
mesh_terms: D000595:Amino Acid Sequence; D061505:Cell Surface Display Techniques; D019976:Cloning, Organism / Q000379:methods*; D032621:DNA Shuffling / Q000379:methods*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D016415:Sequence Alignment
keywords: DNA library; DNA shuffling; directed evolution; phage display recombination cloning