title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.
abstract: We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.
authors.forename: Magnus
authors.initials: M
authors.lastname: Lundqvist
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Edfors
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.
authors.forename: Åsa
authors.initials: Å
authors.lastname: Sivertsson
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.
authors.forename: Björn M
authors.initials: BM
authors.lastname: Hallström
authors.affiliation: KTH - Royal Institute of Technology, Science for Life Laboratory, Stockholm 17165, Sweden.
authors.forename: Elton P
authors.initials: EP
authors.lastname: Hudson
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Tegel
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Holmberg
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden KTH - Royal Institute of Technology, Science for Life Laboratory, Stockholm 17165, Sweden Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2970 Hørsholm, Denmark.
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden johanr@biotech.kth.se.
authors.forename: Johan
authors.initials: J
authors.lastname: Rockberg
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005822:Genetic Vectors; D009693:Nucleic Acid Hybridization
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes