agl5_fig_2.gif (4308 bytes)Schematic diagram of PCR experiments.
750 kanamycin-resistant transgenic lines were produced by Agrobacterium-mediated transformation, and pools of T1 transformants were analyzed using a PCR assay with oligos 1 and 2 to determine if any primary transformants had the desired targeted insertion into AGL5. Oligo 1 is not contained in the KO construct and oligo 2 lies within the kanamycin cassette of the vector. Only a homologous recombination event should yield a product with these two oligos. A single line was identified that appeared to contain the anticipated insertion, and this line was allowed to self. A confirmatory PCR experiment was done on this line using oligos 3 and 2 and the anticipated product was seen. Once we determined that we had segregated away all other T-DNA inserts, PCR reactions using oligos 4 and 5 allowed us to readily identify which plants were homozygous, heterozygous, or wt at the AGL5 locus. These oligos amplify across the genomic region which, in the KO, has lost AGL5 sequence and gained the larger kanamycin cassette.