Figure 4

Solid support rolling circle DNA synthesis from amplified SF-WT90 oligonucleotide nicked with Nt. Alw I. First row: The chemically synthesized oligonucleotide, SF-WT90, was ligated to create a closed circle and incubated with a covalently coupled primer (Amin-L16-Pr SF-WT90 (+) or Amin-L16-Pr SF-WT90 (-)). Following a stringent wash the hybridized circles were amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotides. Second row: The SF-WT90 oligonucleotide, was amplified one round by the method presented in figure 1 (from (+)-strand to (-)-strand). Subsequently, it was ligated to create a closed circle and incubated with a covalently coupled primer (Amin-L16-Pr SF-WT90 (+) or Amin-L16-Pr SF-WT90 (-)). Following a stringent wash the hybridized circles were amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotides. Third row: The SF-WT90 oligonucleotide was amplified two rounds by the method presented in figure 1 (from (+)-strand to (-)-strand and back to (+)-strand). Subsequently, it was ligated to create a closed circle and incubated with a covalently coupled primer (Amin-L16-Pr SF-WT90 (+) primer or Amin-L16-Pr SF-WT90 (-)). Following a stringent wash the hybridized circles were amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotides. Schematic representations indicate the expected outcome of the hybridization events. (+) and (-) indicate the polarity of the probes. The (+)-primer hybridizes to the (+)-probe and the (-)-primer hybridizes to the (-)-probe. Equimolar amounts of probe were applied in each reaction (0.1 μM). Scale bar, 100 μm. At the bottom of the figure a schematic representation of the individual steps in the solid support assay is presented.