Contents - Faperta

468 Biotechnological Approaches for Pest Management and Ecological Sustainability
these to bind to DNA or RNA. If designed properly, these can be used to discriminate
between the right molecule (sequence) and any other DNA/RNA (Holst-Jensen, 2006).
Labeling of molecules with fl uorescence, radioactivity, antibodies, or dyes can also be used
to facilitate the detection of genetically modifi ed foods.
DNA-Based Methods
The DNA-based methods involve detection of the specifi c genes or DNA genetically engineered
into the crop. Although, there are several DNA-based methodologies, the most
commercial testing is conducted using PCR technology. The PCR technique is based on
multiplying a specifi c target DNA, allowing the million- or billion-fold amplifi cation by
two synthetic oligonucleotide primers. The process consists of extraction and purifi cation
of DNA, amplifi cation of the inserted DNA by PCR, and confi rmation of the amplifi ed PCR
product. In principle, PCR can detect a single target molecule in a complex DNA mixture.
The fi rst step involves separation of the two strands of the original DNA molecule. The
fi rst primer matches the start of the coding strand of the DNA, while the second primer
matches the end and the noncoding strand of the DNA to be multiplied. The second step
involves binding of the two primers to their oligonucleotide primers. The third step involves
making two perfect copies of the original double-stranded DNA molecule by adding the
right nucleotides to the end of each primer, using the strands as templates. Once the cycle
is completed, it can be repeated, and in each cycle, the number of copies is doubled, resulting
in an exponential amplifi cation. After 20 cycles, the copy number is 1 million times higher
than at the beginning of the fi rst cycle. The amplifi ed fragment can be detected by gel
electrophoresis or hybridization techniques.
Qualitative PCR Analysis
For general screening purposes, PCR-based methods have been widely used for the detection
of genetically modifi ed crops (Holst-Jensen et al., 2003; Holst-Jensen, 2006). The focus
should be on target sequences that are characteristic for the group to be screened. Genetic
control elements such as the caulifl ower mosaic virus 35S promoter and the Agrobacterium
tumefaciens (Townsend) nos terminator (nos3¢) are present in many genetically modifi ed
crops. The PCR detects the presence of the genetically modifi ed crop, which then needs to
be identifi ed. Primer selection has to be based on target sequences that are characteristic of
individual transgenic crops. The junction sequences between two adjoining DNA segments
can be the target for specifi c detection of the genetic construct such as cross-border regions
between integration site and transformed genetic element of a specifi c transgenic variety or
specifi c sequence alterations. The junction sequences in the integration site (plant-construct
junction fragment) can be used to detect a specifi c transformation event. When the same
gene construct is used to produce different transgenic crops, this will be the only strategy
to distinguish between crops containing the same gene construct. The scheme to test for the
presence of transgenic plants includes initial screening of samples for species-specifi c DNA,
known as housekeeping genes, for example, lectin in soybean or invertase gene (ivrI) in
maize to determine whether DNA from that species can be detected. If DNA is detectable,
samples are then screened using the general genetic elements for the detection of transgenic
crops. Positive indication from initial screening can be followed with screens for specifi c
genes or constructs used in the most common transgenic crops, followed by identifi cation
tests depending on the DNA sample (e.g., cry genes, EPSPS gene, Pat gene, etc.), or more
ideally, for the plant-construct junction fragments.