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Title: Illegitimate recombination in plasmids
Author: Pinder, David
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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Illegitimate recombination mechanisms are important for genetic change within an organism. They are also the cause of many instability problems in biotechnology and have been associated with certain human genetic disorders and cancers. The original aim of this work was to construct a deletion (illegitimate recombination) resistant cosmid based, cloning system, for the cloning of unstable human DNA. Two 'mutant plasmids' pMS5 and pMS7 were isolated. The plasmids were derived from pUC18 and appeared to stabilise the propagation of a long DNA palindrome. The basic concept was to construct a new cosmid using pMS7 as part of the backbone. The construction of two new cosmids cDRII (Deletion Resistant) and cDRIII is described. However, they are unlikely to contain a mutation which stabilises unstable sequences. This thesis also describes the search for the 'mutation' in pMS7 by, single-strand conformation polymorphism and fragment swap analysis. This work led to the isolation of a novel mutation composed of both direct and inverted repeats, which I have called DIR. The presence of DIR in pAC2 (a derivative of pUC18, with the same DNA palindrome as pMS7), supports the absence of a stabilising 'mutation in pMS7. The structure of the DIR mutation is analysed in detail and a hypothesis for its formation is proposed. Finally, the behaviour of four long DNA palindromes (other than that cloned in pMS7), is investigated when ligated into pM* (a derivative of pMS7).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available