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Int J Mol Sci. 2014 Oct 31;15(11):19898-923. doi: 10.3390/ijms151119898.

Mutation scanning in a single and a stacked genetically modified (GM) event by real-time PCR and high resolution melting (HRM) analysis.

Author information

1
Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. sina-elisabeth.ben-ali@ages.at.
2
Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. zmadi@gmx.net.
3
Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. rupert.hochegger@ages.at.
4
Centre for Biosafety-GenØk, PB 6418 Science Park, 9294 Tromsoe, Norway. david.quist@uit.no.
5
Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. bernhard.prewein@ages.at.
6
Department of Nutritional Sciences, University of Vienna, Althanstraße 14, 1090 Vienna, Austria. alexander.haslberger@univie.ac.at.
7
Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria. christian.brandes@ages.at.

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.

PMID:
25365178
PMCID:
PMC4264145
DOI:
10.3390/ijms151119898
[Indexed for MEDLINE]
Free PMC Article

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