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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

DEVELOPING A MOLECULAR TOOL KIT FOR DIAGNOSTIC PCR

Mohamed Moumin, Neima January 2019 (has links)
ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

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