title: Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert.
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 integrated 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.
authors.affiliation: Laboratoire de Méthodologies de la Détection des OGM, Institut National de la Recherche Agronomique, Route de Saint Cyr, Versailles, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Collonnier
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schattner
authors.forename: Georges
authors.initials: G
authors.lastname: Berthier
authors.forename: Francine
authors.initials: F
authors.lastname: Boyer
authors.forename: Géraldine
authors.initials: G
authors.lastname: Coué-Philippe
authors.forename: Annick
authors.initials: A
authors.lastname: Diolez
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Duplan
authors.forename: Sophie
authors.initials: S
authors.lastname: Fernandez
authors.forename: Naïma
authors.initials: N
authors.lastname: Kebdani
authors.forename: André
authors.initials: A
authors.lastname: Kobilinsky
authors.forename: Marcel
authors.initials: M
authors.lastname: Romaniuk
authors.forename: Marc
authors.initials: M
authors.lastname: de Beuckeleer
authors.forename: Marc
authors.initials: M
authors.lastname: de Loose
authors.forename: Pieter
authors.initials: P
authors.lastname: Windels
authors.forename: Yves
authors.initials: Y
authors.lastname: Bertheau
mesh_terms: D024509:3' Flanking Region / Q000235:genetics; D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D005062:European Union; D030841:Food, Genetically Modified; D007881:Legislation, Food; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000737:chemistry; D003313:Zea mays / Q000737:chemistry* / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018744:DNA, Plant