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Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert
Cécile Collonnier1, Alexandra Schattner, Georges Berthier
1Laboratoire de Méthodologies de la Détection des OGM, Institut National de la Recherche Agronomique, Route de Saint Cyr, Versailles, France.
Journal of AOAC International
|April 30, 2005
Summary
The T25 maize transformation event was sequenced, revealing an intact construct integrated into the Huck retrotransposon family. A reliable, event-specific real-time PCR detection system was developed for T25 maize quantitation.
Area of Science:
- Genetics
- Molecular Biology
- Plant Biotechnology
Background:
- T25 is a key maize transformation event authorized in Europe.
- It was developed using polyethylene glycol-mediated transformation with a synthetic pat gene construct.
Purpose of the Study:
- To sequence the T25 insert and characterize its integration site.
- To develop an event-specific detection system for T25 maize.
Main Methods:
- Whole insert sequencing and genome walking strategy.
- Analysis of junction fragments for integration site characterization.
- Development of a real-time PCR assay using Taqman chemistry.
Main Results:
- Confirmed integration of one intact T25 construct copy.
- Identified a truncated 35S promoter at the 5' junction, suggesting rearrangements.
- Integration site showed homology with the Huck retrotransposon family.
- Developed a reliable event-specific detection system for T25 maize.
Conclusions:
- The T25 insert is integrated into the maize genome within a Huck retrotransposon region.
- A robust real-time PCR method is available for specific T25 event quantitation, aiding regulatory compliance.