title: Mutation scanning in a single and a stacked genetically modified (GM) event by real-time PCR and high resolution melting (HRM) analysis.
abstract: Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017×MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found.
authors.affiliation: Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. sina-elisabeth.ben-ali@ages.at.
authors.forename: Sina-Elisabeth
authors.initials: SE
authors.lastname: Ben Ali
authors.affiliation: Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. zmadi@gmx.net.
authors.forename: Zita Erika
authors.initials: ZE
authors.lastname: Madi
authors.affiliation: Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. rupert.hochegger@ages.at.
authors.forename: Rupert
authors.initials: R
authors.lastname: Hochegger
authors.affiliation: Centre for Biosafety-GenØk, PB 6418 Science Park, 9294 Tromsoe, Norway. david.quist@uit.no.
authors.forename: David
authors.initials: D
authors.lastname: Quist
authors.affiliation: Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. bernhard.prewein@ages.at.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Prewein
authors.affiliation: Department of Nutritional Sciences, University of Vienna, Althanstraße 14, 1090 Vienna, Austria. alexander.haslberger@univie.ac.at.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Haslberger
authors.affiliation: Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. christian.brandes@ages.at.
authors.forename: Christian
authors.initials: C
authors.lastname: Brandes
mesh_terms: D020413:3' Untranslated Regions; D001483:Base Sequence; D018744:DNA, Plant / Q000032:analysis*; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D003313:Zea mays / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D018744:DNA, Plant