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Title: Immunochemical detection of mutagenic lesions in DNA
Author: Barlow, Thomas
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Vinyl chloride, a widely used industrial precursor is an established chemical carcinogen. It is metabolically activated to forms which alkylate DNA, producing a range of nucleic acid adducts. Resultant mutagenesis occurs almost exclusively at cytosines which are converted to the exocyclic lesion, 3,N4 ethenodeoxycytidine (C). A monoclonal antibody which specifically recognises C has been developed. The specificity and affinity of this antibody has been determined by ELISA. Immunoaffinity gels have been prepared and an immunoenrichment procedure developed to detect and identify C from normal nucleotides at very low abundance. Immunopurified samples of C have been quantified by CZE on addition of a standard spike with detection sensitivity of 1 C in 108 normal nucleotides. Kinetic studies using this detection system found production of C to be greater in single stranded DNA than in double stranded DNA when incubated with a metabolite of vinyl chloride. Oligonucleotides incorporating C have been synthesised and used in thermodynamic and structural studies to investigate the base pairing effect of C in double stranded DNA. C was found to have a detrimental effect on duplex stability although the overall duplex structure was found to be undistorted. Oligonucleotides labelled with the dansyl group have been synthesised for use as gene probes. A monoclonal antibody with affinity to dansyl was developed to immunochemically detect this reporter group. Femtomole quantities of labelled probe were detected with this system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available