A competitive PCR-based method to detect a single copy of T-DNA insertion in transformants.

Gene function studies benefit from the availability of mutants. In plants, Agrobacterium-mediated genetic transformation is widely used to create mutants. These mutants, also called transformants, contain one or several transfer-DNA (T-DNA) copies in the host genome. Quantifying the copy number of T-DNA in transformants is beneficial to assess the number of mutated genes. Here, we developed a competitive polymerase chain reaction (PCR)-based method to detect a single copy of a T-DNA insertion in transformants. The competitor line BHK- -1 that contains a single copy of competitor BHK- (BHK, Basta, Hygromycin, Kanamycin-resistant genes) was crossed with test transformants and the genomic DNA of F1 plants was subjected to competitive PCR. By analyzing the gray ratio between two PCR products, we were able to determine whether or not the test transformants contained a single copy of T-DNA insertion. We also generated the control lines BHK±1:1 and BHK±2:1 , which contain the target (BHK+ ) and competitor (BHK- ) in a ratio of 1:1 and 2:1, respectively. The ratios of their PCR products are useful references for quantitative analysis. Overall, this method is reliable and simple in experimental manipulations and can be used as a substitute for Southern-blot analysis to identify a single copy of T-DNA insertion in transformants.

TADEA-PCR is a highly efficient method of amplifying unknown flanking fragments of T-DNA transformants.

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.