Figure 4

Improved mapping of the genomic insertion sites in selected transformants using TAIL-PCR. Two additional sets of SAD primers (SAD5–8 and SAD9–12) were synthesized in which one or two extra nucleotides, respectively, were added to the 3′ end in order to reduce their annealing frequency within the S. marinoi genome. Shown are three representative transformants (SM7–8 and SM16) mapped from the promoter end using SAD1–8 (A) and one (SM4) mapped from the both ends using SAD1–12 (B) in which significantly longer PCR products were produced relative to those using the shorter SAD primers. The final PCR products were separated and visualized by gel electrophoresis, with selected individual products then purified and sequenced.