TADEA‐PCR is a highly efficient method of amplifying unknown flanking fragments of T‐DNA transformants
2018
Yang, Yun | Cao, Yuan | Xu, Hua‐Quan | Gao, Long | Guo, Xue | Liu, Xue‐Qiong | Zhang, Li | Zhang, Xue‐Xin | Wang, Dan‐Yang
Forward genetic analysis, widely used to find new gene functions, benefits from the availability of mutants. At present, based on Agrobacterium‐mediated plant transformation technology, many transfer (T)‐DNA transformants have been created. However, cloning their T‐DNA insertion sites, which enables identification of the mutated genes, is still challenging. In this study, we improved adapter ligation‐mediated polymerase chain reaction (A‐PCR), which mainly utilizes the Thermal Asymmetric interlaced reaction and Degenerate sequence‐recognizing restriction Endonucleases (TADE). Using the new method TADE‐mediated A‐PCR (TADEA‐PCR), we successfully cloned 22 of all the 24 junction sites in 10 Arabidopsis thaliana L. transformants that contained 12 T‐DNA insertions in total, giving a success rate of 91.7%. In most cases, the two junction sites resulting from a single T‐DNA insertion were simultaneously cloned. In addition, TADEA‐PCR was able to clone more than two junction sites present in one transformant containing several T‐DNA insertions. Overall, TADEA‐PCR is a powerful technique for cloning T‐DNA insertion sites.
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