Approaches to the cloning of high activity regulatory elements from myeloma cells.
thesisposted on 19.11.2015, 09:07 by Nicholas R. Sutcliffe
Cloning strategies were developed to isolate genomic sequence from the site of plasmid vector integration of myeloma J558L transformants. Two potential approaches were examined. Ligation-mediated anchor PCR was shown to be capable of generating amplification products that included sequence from the integrated plasmid. However, when these were cloned and analysed, they were found to contain various arrangements of primers and plasmid sequence, but no genomic flanking sequence. Further refinement of the procedure failedto generate any products containing flanking sequence. Phage libraries were constructed from transformant genomic DNA. These were screened for clones containing plasmid sequence. A clone was identified, purified and analysed. Although it did contain both plasmid and unknown genomic sequence, its size and arrangement did not tally with information previously gleaned about the integration site and so its significance was hard to establish. Ligation-mediated anchor PCR has been shown to be a potentially quick and convenient cloning method but one which is extremely technically demanding. It is unclear why it failed in this case to generate useful product. Phage cloning is a more laborious approach which did produce results. Although the clone generated was not obviously useful, the potential of the procedure has been demonstrated and it should form the basis of future investigations.