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
- 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?
||Identifiable Products (total 28)
|P-35S*, nos 3'
||26 (or 27)
|P-35S*, nos 3', E9 3', als
|P-35S*, nptII, nos 3'
||26 (or 27)
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