Date

2014

Author

Keskin, Batuhan Birol

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The reliable detection and quantification of Genetically Modified Organisms (GMO) is strongly dependent on validated methods as well as calibration systems. Today, Certified Reference Materials are used as reliable source of template DNA in quantitative real-time PCR assays constructing validated methods for routine analysis of GMOs. In addition to that, obtaining and assessing plasmids for use as real-time PCR standards and positive PCR controls are increasingly used methodologies. To enforce the labeling regulations of GMOs, the application of DNA plasmids as calibrants is becoming essential for the practical quantification of GMOs. This study reports the construction of plasmids for qualitative screening assay for 35S promoter and NOS terminator as GMO elements, and relative quantification assays in Maize and Soya events, Bt11 and GTS 40-3-2 respectively. Reference GM plasmids provided convenient and reliable positive controls for GM PCR tests. Soya GM event GTS 40-3-2 and endogenous control plasmids assessed within-laboratory assays were resulted to be acceptable in terms of reproducibility standard deviation and repeatability relative standard deviation. As a result of verification and measurement uncertainty data based on single laboratory data, constructed plasmids provided an excellent and economic alternative to plant DNA extractions for positive control material. However, further study is needed showing enhanced amplification efficiency and inter-laboratory verification data for using constructed plasmids as template DNA in validated methods.

Subject Keywords

Transgenic organisms., Genetic recombination., Recombinant DNA., Genetic transformation., Polymerase chain reaction.

URI

http://etd.lib.metu.edu.tr/upload/12618140/index.pdf
https://hdl.handle.net/11511/24186

Collections

Graduate School of Natural and Applied Sciences, Thesis