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Updated: Dec 23, 2025
Recombineering Homologous Recombination Constructs in Drosophila
Published on: July 13, 2013
Fusion-PCR generates attL recombination site adaptors and allows Rapid One-Step Gateway (ROG) cloning
Libing Liao1, Lu Yang2, Yanxia Xu2
1Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, 466001, China; Lushan Biotanical Garden, Chinese Academy of Science, Jiujiang, 332900, China; Henan Key Laboratory of Crop Molecular Breeding & Bioreactor, Zhoukou, 466001, China; College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, 466001, China.
Abstract:
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.
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