Analysis of the genetic stability of event NK603 in stacked corn varieties using high-resolution melting (HRM) analysis and Sanger sequencing

The examination of transgenic loci is an integral part of biosafety legislation in the European Union (EU). The EU directive 2001/18/EC requires any genetically modified (GM) event to be stable. Mutations in the nucleotide sequence of GM events must be avoided in the production and use of seeds. In the present work, an F1 and an F2 generation of the corn event NK603 were studied in stacked varieties (NK603 x MON810). The central aspect of this work was to utilize high-resolution melting analysis, real-time PCR, and Sanger sequencing for the examination of genetic stability of the entire construct of NK603 as well as in the regions flanking NK603. To perform such screening, it was necessary to develop specific PCR primers for the NK603 insert. Twenty-five specific primer pairs and PCR reactions were used to screen a total number of 340 samples. In addition to the screening, the NK603 zygosity was determined by a PCR-based testing method. Differences to the published patent sequence occurring in all samples were detected in two locations of the transgenic DNA sequence. These differences were also found in certified NK603 reference material.