Division 1.2.2.2 Information on the sequences actually inserted/deleted

The applicant shall provide the following information:

1. (a)

   the size and copy number of all detectable inserts, both complete and partial; this is typically determined by Southern analysis.

   Probe/restriction enzyme combinations used for this purpose shall provide complete coverage of sequences that could be inserted into the genetically modified plant, such as any parts of the plasmid/vector or any carrier or foreign nucleic acid(s) remaining in the genetically modified plant.

   The Southern analysis shall span the entire transgenic locus(i) as well as flanking sequences and include all appropriate controls.

   For the determination of copy number of the insert, complementary methods may also be used (such as real-time PCR);

2. (b)

   the organisation and sequence of the inserted genetic material at each insertion site in a standardised electronic format, with the aim of identifying changes in the inserted sequences compared to the sequence intended for insertion;

3. (c)

   in the case of deletion(s), size and function of the deleted region(s), whenever possible;

4. (d)

   subcellular location(s) of insert(s) (nucleus, chloroplasts, mitochondria, or maintained in a non-integrated form) and methods for its/their determination;

5. (e)

   sequence information in a standardised electronic format for both 5’ and 3’ flanking regions at each insertion site, with the aim of identifying interruptions of known genes.

   Bioinformatic analyses shall be conducted using up-to-date databases with the aim of performing both intraspecies and interspecies similarity searches.

   In the case of genetically modified plants containing stacked transformation events, the safety of potential interactions between any unintended modifications at each insertion site shall be assessed;

6. (f)

   Open Reading Frames (hereafter referred to as ‘ORFs’ and defined as any nucleotide sequence that contains a string of codons that is uninterrupted by the presence of a stop codon in the same reading frame) created as a result of the genetic modification either at the junction sites with genomic DNA or due to internal rearrangements of the insert(s).

   The ORFs shall be analysed between stop codons, not limiting their lengths. Bioinformatic analyses shall be conducted to investigate possible similarities with known toxins or allergens using up-to-date databases.

   The characteristics and versions of the databases shall be provided.

   Depending on the information gathered, further analyses (such as transcription analysis) may be needed to complete the risk assessment.