The 2,4-dinitrophenyl (DNP) group is an example of a hapten with potential use as a non-radioactive labelling group for oligonucleotides. This label is highly antigenic, inexpensive, chemically simple, sterically undemanding, and is not found <I>in vivo</I>. A series of non-nucleoside-based DNP phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid phase synthesis. Oligonucleotides labelled in this way were synthesised in very high yield and easily purified by reversed-phase HPLC. The lengths of spacer arms between the DNP label and the oligonucleotide phosphate backbone have been varied in order to determine the optimum length for anti-DNP antibody binding. The optimum number of DNP labels for maximum signal strength is also reported. The labelled oligonucleotides were detected using standard ELISA methodology, employing a monoclonal IgG mouse anti-DNP antibody, giving sensitivities equivalent to those obtainable in the visualisation of biotinylated oligonucleotides. DNP labelled phosphoramidite and triphosphate derivatives of 2'-deoxyuridine have also been synthesized and used to label oligonucleotides. As an alternative to the above DNP based non-radioactive labelling system, single and multiple dansyl based phosphoramidites have been synthesized and used to fluorescently label oligonucleotides. Protein-dansyl conjugates have been prepared for the production of anti-dansyl antibodies which would allow the immunogenic detection of this label. Oligonucleotide probes attached directly to alkaline phosphatase have been prepared using inexpensive, commercially available reagents and used in hybridisation experiments to detect 0.3fmoles of target DNA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:651853 |
| Date | January 1994 |
| Creators | Grzybowski, John |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12069 |
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