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Title: Development of immunochemical methods to detect point mutations in the form of mismatched base pairs in DNA
Author: Turner, Paul C.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Immunoanalytical methods, based on monoclonal antibodies may prove effective in detecting and enriching mismatches. This has been investigated. A series of 15mer single stranded oligonucleotide of defined sequence linked chemically by means of a tetraethylene glycol linker to a complementary 15mer strand were synthesised in such a way that the intramolecular duplex contains a specific central mismatched based pair. Since the two strands were chemically linked, a highly stable duplex, indicated by UV melting studies, was formed which should persist in vivo. These synthetic duplexes were used to evoke an immune response by direct immunisation, or as methylated BSA complexes. In a related approach a series of 75mer duplexes have been constructed such that they contain four evenly spaced mismatched base pairs. Each sequence was extended by the addition of a 5mer phosphorothioate oligonucleotide at the 3' end to protect the DNA from nuclease degradation. In a third approach, a C100mer phosphorothioate sequence was produced. Antibodies recognising cytosine in C100mer may cross react with mismatches containing this base (A:C; C:C; T:C). Hybridoma cell lines obtained after fusions from 15mer duplex immunised mice were screened for antibody production using the ELISAs initially developed. These experiments failed to produce antibodies recognising mismatched base pairs. An ELISA format in which biotin labelled oligonucleotide duplexes were immobilised on streptavidin coated plates was used as a solid phase. For immunisations with the larger duplexes the improved screening methods were used as a solid phase. For immunisations with the larger duplexes the improved screening methods were used to monitor antibody production. Although anti-mismatch antibodies were detected using AC75 and GT75 no monoclonal cell lines were isolated. The C100mer produced antibodies that bound strongly to single stranded DNA containing cytosine, and cross reacted with duplexes containing AC and CC mismatches. Monoclonal cell lines were isolated from these experiments with retained antibody specificity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available