Triprimer-PCR method: rapid and reliable detection of transgenes in transgenic rice plants.

We designed a triprimer-PCR system for detection of transgenes and applied for analysis of two different kinds of transgenic rice plants, which were previously transformed with the plasmids pBY605RR or pARP7 containing a maize ribosome-inactivating protein gene, Zmcrip3a, and a herbicide-resistant gene, bar. Genomic Southern-blot analysis demonstrated that the transgenes were stably inherited to their R1 progenies without changes in configuration. The resulting data were used as a reference for triprimer-PCR analysis. The triprimer-PCR system uses an endogenous gene as an internal standard which shares an identical priming site for one primer with a transgene while each of the other two primers is specific to either the transgene or the endogenous gene. Triprimer-PCR analysis was carried out on genomic DNA isolated from 24 different progenies of the pBY605RR- and the pARP7-transformed lines that contain different copy numbers of transgenes. The RbcS:Zmcrip3a junction region of the pBY605RR integrated in rice chromosomes, together with the endogenous RbcS, was efficiently amplified, producing 440 and 250 bp expected PCR products. Also, the Act1:Zmcrip3a junction region of the transgene pARP7 with the endogenous Act1 was similarly amplified, producing 540 and 340 bp expected PCR products. The two PCR products in each set of experiments were observed consistently and independently of copy numbers or rearrangements of the transgene. Thus, the triprimer-PCR strategy may provide a rapid and reliable method for confirming transformation or analyzing segregation of transgenes at the molecular level.