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Title: Optimisation and development of the post-labelling assay
Author: Spencer-Fry, J. E.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 2001
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The research contained within this thesis has a strong technical bias and is centred around developing novel procedures and techniques to move the postlabelling assay from its current status to a safer, more reliable and reproducible procedure consistently producing good quality data. In meeting these study objectives the postlabelling assay would not only be an important biomarker in monitoring exposure to environmental genotoxic compounds but it would provide a reliable biomarker for such monitoring, something not previously achieved. For the study the assay procedure was broken down into six stages; sample preparation, DNA digestion, adduct enrichment, radioactive labelling, adduct resolution and adduct quantification. Each stage in the assay was scrutinised, potential improvements identified, conditions optimised for maximum performance and reliability and suitable assay controls prepared and introduced to evaluate the impact of such changes on the performance criteria of the assay. Where appropriate, novel techniques such as the use of minicolumns for adduct enrichment and the use of alternative isotopes such as [γ33P]ATP were developed and combined with other assay modifications to improve safety, enhance reliability in a bid to reduce the complexity of the procedure and tackle the labour intensity of the approach. As a result of the research performed in this study the laboratory was asked to participate in an "Interlaboratory Trial for the Standardisation and Validation of DNA Adduct Postlabelling Methods". The trial provided an ideal opportunity to evaluate the improved assay procedure developed within this study alongside the protocol recommended by the trial co-ordinators. Many of the trial findings and recommendations correlated with those identified and reported in this thesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available