Solid-phase cloning for high-throughput assembly of single and multiple DNA parts.
Bottom Line: 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 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%.
Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.Show MeSH
Mentions: Magnetic solid-phase support enables easy DNA capture, washing and switching to preferred reaction conditions between assembly steps. The procedures of the two methods are outlined in Figure 1. The restriction based method (Figure 1A) uses the paramagnetic beads to purify and sequentially wash, buffer exchange and digest PCR products, thereby providing a very simple and robust workflow for generation of ligated constructs without need for manual gel or spin-column based purification steps. Briefly, RE-based SPC starts by capturing PCR amplified inserts on streptavidin-coated paramagnetic beads, which then are sequentially washed, buffer-exchanged and incubated at appropriate temperatures for serial solid-phase digestions using independent REases in a liquid handler.
Affiliation: KTH-Royal Institute of Technology, School of Biotechnology, AlbaNova University Center, Stockholm 10691, Sweden.