TADEA-PCR is a highly efficient method of amplifying unknown flanking fragments of T-DNA transformants
| Autor: | Dan-Yang Wang, Li Zhang, Xue-Qiong Liu, Long Gao, Yuan Cao, Hua-Quan Xu, Zhang Xuexin, Xue Guo, Yang Yun |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
DNA
Bacterial 0106 biological sciences 0301 basic medicine Physiology Mutant Plant Science Biology Polymerase Chain Reaction 01 natural sciences Genetic analysis law.invention 03 medical and health sciences chemistry.chemical_compound Adapter (genetics) law Genetics Gene Polymerase chain reaction Cloning Sequence Analysis DNA Cell Biology General Medicine Restriction enzyme 030104 developmental biology chemistry Mutation DNA 010606 plant biology & botany |
| Zdroj: | Physiologia Plantarum. 164:242-250 |
| ISSN: | 0031-9317 |
| DOI: | 10.1111/ppl.12681 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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