Figure 4

Agarose gel electrophoresis of TAIL-PCR products. A: TAIL-PCR (secondary and tertiary reactions are shown) from one putative transgenic line with four different arbitrary primers together with wild type control with one arbitrary primer. Only AD3 primer produced specific product recognizable from the slight shift in size due to the nested LB priming at the T-DNA junction site. Other arbitrary primers did not produce clear, specific products; only faint non-specific bands also present in the wild type control are seen, as exemplified by AD7 primer. B: TAIL-PCR reactions with the same arbitrary primer (AD7) on three putative transgenic plants and a wild type control, all three TAIL-PCR reactions shown. The primary TAIL-PCR reaction produces only small amount of the specific product, not yet visible on the gel, and the specific products start to emerge at the secondary reactions. Two of the three samples shown here proved to originate from the same gene transfer event (J47/1), and one showed a different product pattern, thus being a different event (J47/2). No specific products are shown in the wild type control. Strawberry clones: J47/1–3; WT, wild type. Arbitrary primers: AD2, AD3, AD6, AD7. TAIL-PCR reactions: I, primary (LB1 primer); II, secondary (LB2 primer); III, tertiary (LB3 primer). Specific TAIL-PCR reaction products excised from gel for sequencing are marked with blue oval. C: Position of T-DNA junction site specific primers at the left T-DNA border region of the pCAMBIA 1391Z gene transfer vector. The three nested T-DNA left border specific primers were designed for the TAIL-PCR analysis. The vector sequence is shown on uppercase and the left T-DNA border region as orange colour.