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Characterization and Event Specific-Detection by Quantitative Real-Time PCR of T25 Maize Insert

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Abstract:

T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5′ junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5′ end of the integrat ed DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman® chemistry.

Document Type: Research Article

Affiliations: 1: Laboratoire de Méthodologies de la Détection des OGM, Institut National de la Recherche Agronomique, Route de Saint Cyr, 78206 Cedex Versailles, France. 2: Laboratoire de Biométrie et Intelligence Artificielle UR341, Domaine de Vilvert, Jouy-en-Josas Cedex 78352, France. 3: Molecular and Biochemical Analytical Services, Bayer BioScience NV, J. Plateaustraat 22, B-9000 Ghent, Belgium. 4: Department of Plant Genetics and Breeding, Centre for Agricultural Research, Caritasstraat 21, 9090 Melle, Belgium.

Publication date: March 1, 2005

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  • The Journal of AOAC INTERNATIONAL publishes refereed papers and reviews in the fields of chemical, biological and toxicological analytical chemistry for the purpose of showcasing the most precise, accurate and sensitive methods for analysis of foods, food additives, supplements and contaminants, cosmetics, drugs, toxins, hazardous substances, pesticides, feeds, fertilizers and the environment available at that point in time. The scope of the Journal includes unpublished original research describing new analytical methods, techniques and applications; improved approaches to sampling, both in the field and the laboratory; better methods of preparing samples for analysis; collaborative studies substantiating the performance of a given method; statistical techniques for evaluating data. The Journal will also publish other articles of general interest to its audience, e.g., technical communications; cautionary notes; comments on techniques, apparatus, and reagents.
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