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Title: p53 mutations as a marker of malignancy in breast and bladder cancer
Author: Phillips, Hamish Andrew
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Mutations of the p53 gene are common in a wide variety of human malignancies. Detection of such mutations opens up the possibility of their use as a marker for the presence of malignancy, with potential applications in screening, diagnosis and follow up. This project investigated this potential. Single Stranded Conformational Polymorphism (SSCP), a PCR based gel electrophoresis technique, was used to screen for p53 mutations in bladder washing samples from patients with bladder cancer and breast fluid samples obtained by nipple aspiration from patients with breast cancer. Exons 5-8 of the p53 gene which contain over 80% of reported mutations were examined individually. The SSCP assay was optimised using DNA extracted from 6 cancer cell lines (2 with normal p53 and the others each with a mutation in 1 of exons 5,6,7 or 8) using 6 sets of gel conditions. Each mutant carrying cell line could be identified by an unambiguously abnormal SSCP pattern under at least 3 tests of gel conditions. Dilutions of each of the mutant cell lines were made with wild type cells to determine the sensitivity of SSCP to detect the mutation in DNA extracted from the mixture of cells. (This was of fundamental importance as the clinical samples studied would inevitably contain such a mixture). SSCP was performed for each dilution series using gel conditions able to demonstrate that particular mutation. The sensitivity in detecting mutations varied (range 1-30%) and was dependent on the conditions used. Using optimal conditions for each mutation a distinct extra band corresponding to the mutation was discernable at the 1 in 100 level of exon 8 and at 1 in 20 for exons 5,6 and 7. These results illustrate the importance of gel conditions in determining the sensitivity of SSCP in detecting mutations in mixed cell populations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available