Figure 6

Comparison of the chemically synthesized oligonucleotide WT90-66b and the oligonucleotide contained within the suicide cassette in SF-WT90 following amplification in a solid support rolling circle DNA synthesis assay. The chemically synthesized padlock probe WT90-66b (left) and SF-WT90 (amplified two rounds (from (+)-strand to (-)-strand and back to (+)-strand) and cleaved with Mly I; the probe was purified by PAGE after each round) (right) were incubated with a covalently coupled primer (Amin-L16-Mly I (+)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of the ID 16 detection oligonucleotide. Equimolar amounts of probe were applied in each reaction (0.1 nM). Scale bar, 100 μm.