Fusion-PCR generates attL recombination site adaptors and allows Rapid One-Step Gateway (ROG) cloning.

Gateway recombination-based cloning, which eliminates the use of restriction endonucleases and ligase, has been widely used for the construction of high-throughput (HTP) vectors. However, this approach is very expensive and its two-stage reaction process is laborious and time consuming. Therefore, we developed a Gateway cloning method that uses fusion-PCR to generate attL recombination site adaptors, and the PCR products, which can be directly cloned into destination vectors, giving rise to Rapid One-Step Gateway (ROG) Cloning. 100% of cloning efficiencies were obtained by this ROG method. This method has no BP reaction/entry clone step, thus halving the cost and time consumed. Overall, this work provides a highly efficient, rapid, low-cost method for directional recombination cloning.