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Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae

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dc.contributor.author Anderson, Peter. en_US
dc.date.accessioned 2009-07-09T18:38:55Z
dc.date.available 2009-07-09T18:38:55Z
dc.date.issued 2003-07-09T18:38:55Z
dc.identifier.uri http://hdl.handle.net/10464/1945
dc.description.abstract The manipulation of large (>10 kb) plasmid systems amplifies problems common to traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are uncommon and very rarely located in opportunistic locations. Making site-specific deletions and insertions in larger plasmids consequently leads to multiple step cloning strategies that are often limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies. Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are systems that typify such situations. Recombinational cloning techniques based on homologous recombination in Saccharomyces cerevisiae that circumvent many ofthese common problems have been developed. However, these techniques are rarely realistic options for such large plasmid systems due to the above mentioned difficulties associated with the addition ofrequired yeast DNA replication, partitioning and selectable marker sequences. To determine ifrecombinational cloning techniques could be modified to simplify the manipulation of such a large plasmid system, a recombinational cloning system for the creation of human adenovirus EI-deletion rescue plasmids was developed. Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone sufficient to foster successful recombinational cloning without additional partitioning sequences, using only slight modifications of existing protocols. In addition, we describe conditions for efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2 kb) and insertion of donor fragment DNA using only a single non-unique restriction site. The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and positive selection to reduce background transformants to undetectable levels. The modification of existing protocols as described in this report facilitates the use of recombinational cloning strategies that are otherwise difficult or impractical for use with large plasmid systems. Improvement of general recombinational cloning strategies and strategies specific to the manipulation ofthe adenovirus genome are considered in light of data presented herein. en_US
dc.language.iso eng en_US
dc.publisher Brock University en_US
dc.subject Adenoviruses. en_US
dc.subject Plasmids. en_US
dc.subject Cloning. en_US
dc.subject Genetic recombination. en_US
dc.subject Saccharomyces cerevisiae. en_US
dc.title Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae en_US
dc.type Electronic Thesis or Dissertation en_US
dc.degree.name M.Sc. Biological Sciences en_US
dc.degree.level Masters en_US
dc.contributor.department Department of Biological Sciences en_US
dc.degree.discipline Faculty of Mathematics and Science en_US


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