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TADEA‐PCR is a highly efficient method of amplifying unknown flanking fragments of T‐DNA transformants
Author(s) -
Yang Yun,
Cao Yuan,
Xu HuaQuan,
Gao Long,
Guo Xue,
Liu XueQiong,
Zhang Li,
Zhang XueXin,
Wang DanYang
Publication year - 2018
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/ppl.12681
Subject(s) - biology , microbiology and biotechnology , cloning (programming) , transformation (genetics) , polymerase chain reaction , dna , genetics , adapter (computing) , gene , inverse polymerase chain reaction , primer dimer , agrobacterium , homing endonuclease , multiplex polymerase chain reaction , endonuclease , computer science , electrical engineering , programming language , engineering
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.