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

Abstract

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.

A prolific and robust whole‐genome genotyping method using PCR amplification via primer‐template mismatched annealing

“…We previously developed the flanking sequence tag (FST)‐seq method for high‐efficiency isolation of unknown sequences flanking a known DNA fragment (Chang et al, 2018). The FST‐seq method uses specific nested primers to anneal to the known target sequence and adapters that are added by Tn5 transposase.…”

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

“…To facilitate the subsequent use of BHK − ‐1 , we cloned the insertion sites of T‐DNA through TADEA‐PCR (Yang et al, 2018; Figure 5D). PCR amplification confirmed this insertion, and long‐range PCR produced a single band with the expected molecular size (Figure 5G).…”

“…After washing twice with 70% alcohol, the DNA was dissolved in 30 μl Tris-EDTA (TE) buffer. The dissolved DNA was either used as the template for PCR or subjected to endonuclease digestion for TADEA-PCR as previously described (Yang et al, 2018).…”

“…Theoretically, a saturated mutant library in which every gene has a corresponding T‐DNA‐inactivated variant can be established if the transformants are sufficient in number. Now, some methods, for example inverse PCR (Ochman et al, 1988), hiTAIL‐PCR (Liu & Chen, 2007), and TADEA‐PCR (Yang et al, 2018), have been developed to identify the mutated genes through cloning the insertion sites of T‐DNA in the host genome, which further benefits the use of transformants in genetic studies.…”