A simple method for identifying plant/T-DNA junction sequences resulting from Agrobacterium-mediated DNA transformation.

摘要

Analysis of the junction sequences spanning the region between the T-DNA borders and genomic DNA after Agrobacterium-mediated transformation gives a clear demonstration of genomic integration. Procedures available for border junction analysis (plasmid rescue, random primed PCR and inverse PCR) can be problematic, so a simplified method was developed for plants transformed by A. tumefaciens harbouring the binary vector pBI121. DNA was isolated from transgenic tobacco plants, digested by a TaqI restriction enzyme and ligated into an ACCI-digested pUC18 plasmid. The ligation mixture was then subjected to PCR amplification using one of the PUC universal primers and another primer which pairs with the T-DNA sequence between the TaqI site and the right(RB) or left border (LB). The PCR products can then be separated on an agarose gel, sequenced and compared with sequences adjacent to the RB and LB of the transforming binary vector.