Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning

Soili I Lehtonen; Barbara Taskinen; Elina Ojala; Sampo Kukkurainen; Rolle Rahikainen; Tiina A Riihimäki; Olli H Laitinen; Markku S Kulomaa; Vesa P Hytönen

doi:10.1093/protein/gzu050

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning

Protein Eng Des Sel. 2015 Jan;28(1):23-8. doi: 10.1093/protein/gzu050. Epub 2014 Dec 1.

Authors

Soili I Lehtonen¹, Barbara Taskinen², Elina Ojala³, Sampo Kukkurainen², Rolle Rahikainen², Tiina A Riihimäki¹, Olli H Laitinen⁴, Markku S Kulomaa⁵, Vesa P Hytönen⁶

Affiliations

¹ BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland.
² BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Fimlab Laboratories, Biokatu 4, FI-33520 Tampere, Finland.
³ BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Next Biomed Technologies NBT Oy, Viikinkaari 6, FI-00790 Helsinki, Finland.
⁴ The Center for Infectious Medicine, Department of Medicine HS, Karolinska Institute, Karolinska University Hospital, Huddinge F59, SE-141 86 Stockholm, Sweden.
⁵ BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Tampere University Hospital, FI-33521 Tampere, Finland.
⁶ BioMediTech, FI-33014 University of Tampere, Biokatu 6, Finland Fimlab Laboratories, Biokatu 4, FI-33520 Tampere, Finland vesa.hytonen@uta.fi.

PMID: 25445152
DOI: 10.1093/protein/gzu050

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.

Keywords: DNA library; DNA shuffling; directed evolution; phage display recombination cloning.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Cell Surface Display Techniques
Cloning, Organism / methods*
DNA Shuffling / methods*
Directed Molecular Evolution
Escherichia coli / genetics
Gene Library*
Molecular Sequence Data
Sequence Alignment