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

Libing Liao; Lu Yang; Yanxia Xu; Xiaoli Li; Guangxuan Tan; Beibei Fu; Kelei Duan; Zhiqiang Li; Deshui Yu; Chengwei Li

doi:10.1016/j.biochi.2020.04.002

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

Biochimie. 2020 Jul:174:69-73. doi: 10.1016/j.biochi.2020.04.002. Epub 2020 Apr 20.

Authors

Libing Liao¹, Lu Yang², Yanxia Xu², Xiaoli Li³, Guangxuan Tan³, Beibei Fu², Kelei Duan², Zhiqiang Li², Deshui Yu⁴, Chengwei Li⁵

Affiliations

¹ Key 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.
² Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, 466001, China; Henan Key Laboratory of Crop Molecular Breeding & Bioreactor, Zhoukou, 466001, China; College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, 466001, China.
³ Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, 466001, China; Henan Key Laboratory of Crop Molecular Breeding & Bioreactor, Zhoukou, 466001, China.
⁴ Key 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. Electronic address: [email protected].
⁵ Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, 466001, China; Henan Key Laboratory of Crop Molecular Breeding & Bioreactor, Zhoukou, 466001, China; Henan Engineering Research Center of Crop Genome Editing, Henan Institute of Science and Technology, Xinxiang, 453003, China. Electronic address: [email protected].

PMID: 32325113
DOI: 10.1016/j.biochi.2020.04.002

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.

Keywords: Agrobacterium-mediated transient expression assay; Fusion-PCR; Rapid One-Step Gateway (ROG) cloning.

MeSH terms

Agrobacterium tumefaciens / genetics
Cloning, Molecular / methods*
Genetic Vectors*
Nicotiana / genetics
Plant Proteins / biosynthesis
Polymerase Chain Reaction / methods*
Recombination, Genetic

Substances

Plant Proteins