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4.2 Development of screening methods

The promoter from cauliflower mosaic virus is the most abundantly used transgenic element in approved genetically engineered crops; it is present in 22 out of 28 plants in its original version or as one of its variants (see Promoters in approved genetically modified agricultural crops). At least six different publications with respect to the origin of the respective P-35S derivative used in the various crops have been cited in publicly available petition documents (Franck et al., 1980; Gardner et al., 1981; Odell et al., 1985; Kay et al., 1985; Pietrzak et al., 1986; Sanders et al., 1987). Some of the versions mentioned display only minor differences or vary by the fusion to distinct 5'-untranslated regulatory regions (Promoters in approved genetically modified agricultural crops). It will be necessary to take these differences into account for the development of broadly applicable screening methods. A PCR-assay based on P-35S sequences that have been shown to be present in various transgenic crops has already been developed (Pietsch et al., 1997; Waiblinger et al., 1997).

Nos 3', originally derived from Agrobacterium tumefaciens (Hernalsteens et al., 1980; Depicker et al., 1982; Bevan et al., 1983; Bevan, 1984; An, 1986), is the most frequently used terminator in approved transgenic crops, present in at least 16 out of 28 approved products (Approved genetically modified crops in the United States, Approved genetically modified crops in the European Community and Terminators in approved transgenic agricultural crops). A PCR-assay based on nos 3' sequences has already been developed and tested (Pietsch et al., 1997; Waiblinger et al., 1997). At least four of the six genetically engineered crops that are not detectable on the basis of P-35S sequences should be detectable by a PCR assay using nos 3' sequences (Tables 3 and 5). The two products that can be assayed neither by a PCR test based on P-35S nor on nos 3' sequences are genetically modified oilseed rape from Monsanto (ID 65) and cotton developed by DuPont (ID 22). A PCR assay for the gene for acetolactate synthase (als) that is present in this cotton has been described already (Petition from DuPont, 1995). Detection methods for the oilseed rape from Monsanto may be developed on the basis of the described genetic elements (Approved genetically modified crops in the United States).

For a PCR screening method to be widely applicable, the following criteria should be met:

• Primers should be selected that are specific for genetic elements present in a large number of genetically engineered agricultural crops.
• The genetic elements on which the assay is based should not occur naturally in the respective plants.
• The assay should not rely on genetic elements that occur in organisms that may appear frequently as contaminants of the food stuff under analysis.
• Within the chosen elements, sequences should be selected that allow specific identification of as many variants as possible of the respective element. In addition, primers with complementary sequences or primers with a secondary structure should be avoided.
• The designed amplicon should be relatively small to warrant broad applicability of the test also to heat-treated samples or materials with low pH and/or highly degraded DNA. In this respect, amplified fragments should not exceed 500 basepairs; a length between 150 and 300 basepairs seems ideal. An even smaller amplicon length (below approximately 100 basepairs) is possible; however, it may require the separation of the amplification products in special type of gels in order to distinguish frequently appearing artificial PCR-products of twice the length of the primers (in general 40-50 (2 x 20-25) basepairs) from the actual target sequence.

Table 19: How many PCR systems are needed?

P-35S* = P-35S including derivatives

Apart from the combination P-35S*/nos 3' there are a number of other combinations that would permit screening for the presence of most approved genetically engineered crops, some of which are described in Table 19. Other combinations are possible by including data from the Approved genetically modified crops in the United States, Approved genetically modified crops in the European Community, Structural genes introduced into approved transgenic crops, Promoters in approved genetically modified agricultural crops and Terminators in approved transgenic agricultural crops such that the identification of products from all approved transgenic crops is achievable.

Screening methods using P-35S and nos 3' sequences evidently are the most favourable candidates for broad method applicability. Assays based on nptII sequences may also be promising, although it has to be taken into consideration that nptII is frequently found in bacteria (Smalla et al., 1993; Redenbaugh et al., 1994) and therefore might lead to false-positive results. In order to judge the reliability of positive results from screening methods based on P-35S and nos 3' sequences, it might also be worthwhile to assess the probability of naturally occurring contamination of foods by plant pest organisms, such as cauliflower mosaic virus or Agrobacterium.