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Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts

Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts

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ODRONITZ, Florian, 2004. Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts [Master thesis]

@mastersthesis{Odronitz2004Estab-8134, title={Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts}, year={2004}, author={Odronitz, Florian} }

2004 2011-03-24T17:40:52Z In the work presented, I was able to characterise several aspects of an in vitro DNA replication system with human cell extracts.<br />I could confirm that plasmids without special sequence characteristics are replicated by the system and that the replication of each template takes place only once, resembling the way genomic DNA is replicated in vivo. The occurrence of different kinds of replication intermediates was shown by electron microscopy, and the fate of the template DNA during the reaction was clarified. I also demonstrated that only a specific form of DNA can serve as substrate and that the products of the reaction can be separated by differential digest in a reasonable manner. Furthermore I was able to proof that the factors responsible for replication in vitro are the the same ones driving the reaction in the cell. It could also be demonstrated that the efficiency of the reaction depends on the cell cycle stage of the cells the protein extracts were prepared from. At last the reaction could be inhibited by depletion of ORC proteins, although the inhibition could not be reverted by the addition of recombinantly expressed ORC complexes.<br />In conclusion this work is a contribution towards the complete characterisation of an in vitro replication assay as a model for the replication of the genome in human cells. Odronitz, Florian Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts eng deposit-license 2011-03-24T17:40:52Z Odronitz, Florian application/pdf

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

F_Odronitz_Diplomarbeit.pdf 124

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