A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis-4

Copyright information:Taken from "A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis"http://www.biomedcentral.com/1472-6750/7/49BMC Biotechnology 2007;7():49-49.Published online 16 Aug 2007PMCID:PMC2040145.16-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 either the ID 16 or the anti ID 16 detection oligonucleotide. : The SF-WT90 oligonucleotide, was amplified one round by the method presented in figure 1 (from (+)-strand to (-)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) or 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 either the ID 16 or the anti ID 16 detection oligonucleotide. : The SF-WT90oligonucleotide was amplified two rounds by the method presented in figure 1 (from (+)-strand to (-)-strand and back to (+)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or 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 either the ID 16 or the anti ID 16 detection oligonucleotide. 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 is a schematic representation of the individual steps in the solid support assay.