Jones, Christopher; (1998) Enzymatic assays and pcr-based dna fingerprinting in mutation detection. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Enzymatic Assays and PCR-based DNA Fingerprinting in Mutation Detection The detection of chemical mutagens by in vitro tests using bacterial and mammalian cell lines relies on alterations in a particular phenotype which can be traced to specific changes in the governing genes. Only a small target gene is investigated; mutations in the majority of the genome have no effect on the phenotype and are not detected. In order to overcome this, the potential of a number of different genotypic mutation assays were investigated. Firstly, isolated DNA in vitro was exposed to the known mutagens UV light and cis- DDP; the effects of the resultant covalent DNA adducts formed on the activity of nucleases were examined. Greatest sensitivity was achieved with the indiscriminantly-cutting DNase I on plasmid and lambda DNA. However, despite the speed, cost-effectiveness and ease using this system, the level of sensitivity is two to three orders of magnitude lower than that routinely achieved using instrumental analysis. Secondly, Randomly Amplified Polymorphic DNA (RAPD) fingerprinting was utilised to investigate mutations caused by genetic disease, and by exposing bacterial and human cells to UV light. Under optimised conditions, reproducible banding patterns may be achieved for a given arbitrarily-chosen primer and template DNA. Any mutation that occurs in the template genomic DNA such that a) a primer is no longer able to bind; b) a new primer binding site is formed; or c) two distant binding sites are brought to within an amplifiable distance of each other, will lead to an alteration of the mutant fingerprint as compared to the control. The major drawbacks of this relatively simple technique is the inability to characterise the mutation, as well as the difficulty of detecting mutant DNA within a large population of non-mutants. Finally an attempt was made to characterise some of the mutations observed with the RAPD fingerprinting experiments using restriction enzyme cleavage, multiplex PCR, SSCP, chemical and enzymatic mismatch cleavage.

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