Detection of Genetically Altered Corn and Soybean Food Products by Polymerase Chain Reaction

1
Detection of Genetically Altered Corn and Soybean
Food Products by Polymerase Chain Reaction

• Consuelo J. Alvarez, Adrienne R. Hampton and Sara
  K. Lee
• Department of Natural Sciences, Longwood
  University Farmville, VA 23909 USA

Background
Data Analysis
Genetically Modified (GM) crops have been
engineered to facilitate the expression of
desirable traits such as pesticide resistance,
herbicide resistance or increased nutritional
value. These modifications are achieved through
the insertion of one or more specific bacterial
genes into the genomes of target agricultural
crops like corn, soy, cotton, wheat, squash,
tomatoes, canola, papaya, and potatoes. In the
United States, around 30-50 of the total land
devoted to growing corn, soybeans and cotton uses
genetically modified versions. In the 1990s
USDA approved CRY1Ab genetically modified corn
seeds and CP4-EPSPS genetically modified soybean
seeds to be used in human food as well as in
animal feed. Other genetic modifications are
restricted to animal feed only, for example CRY9
in corn. Product labels, however, are not
required to inform potential consumers that the
food contains genetically modified components.
Though no allergic reactions to CRY1Ab or
CP4-EPSPS proteins have been reported, concerns
have arisen regarding regulatory measures applied
to genetically modified foods and has become the
focus of novel investigations. CRY is short
for Crystal gene/protein, and it is naturally
found in the bacterium Bacillus thuringiensis.
CRY genes code for CRY d-endotoxin proteins with
pesticide properties. The most common corn pests
are the European corn borer (ECB, Ostrinia
nubilalis), the Southwestern corn borer (SWCB,
Diatraea grandiosella) and the pink borer
(Sesamia cretica) which cause billions of dollars
of damage to crops each year. Since a truncated
version of CRY1Ab containing only the active
toxin-encoding fragments of the gene is used for
transformation in corn (YieldGardR ), promoters
are added to enhance CRY protein expression. The
outcome of the presence of this modification has
reduced and even eliminated the need for
pesticide application on some crops. CRY protein
has 3 domains (Figure 1). Domain I is involved
in the formation of an ion-channel that
contributes to the perforation of the insects
midgut (this eventually kills the insect).
Domain II participates in the midgut cell
receptor recognition and binding. And domain III
has been reported to be involved in ion-channel
activity, receptor binding and structural
stability.
Intact genomic DNA presence was established when
a fragment was seen around the 23 K base pairs
markers of l-Hind III/fX174 Hae III DNA ladder.
A 0.8 agarose gel prepared in TAE (Tris.HCl,
Acetic acid, EDTA) buffer was run and DNA was
visualized under UV light after staining with
ethidium bromide (Figure 5). PCR products for
corn samples (Figure 6) and PCR products for soy
samples (Figure 7) were identified in a 2.0
agarose gel prepared in TAE (Tris.HCl, Acetic
acid, EDTA) buffer and DNA was visualized under
UV light after staining with ethidium bromide.
Their sizes were compared to the 100 base pairs
ladder of New England Bio-Labs. See their
respectively figure legend for specific sizes.
Figure 3. Chemical synthesis of
5-Enolpyruvylshikimate-3 Phosphate (EPSP) from
Phosphoenol Pyruvate (PEP) and Shikimate3-Phosphat
e (S3P).
Research Question
Do any locally available foods contain CRY1Ab
and/or CP4-EPSPS genetic modifications?
Goals

• Assess food products containing corn for the
  presence of CRY1Ab modifications.
• Assess food products containing soy for the
  presence of CP4-EPSPS modifications.
• Compile a data bank of food products from
  local markets containing the above genetic
• modifications.
• Quantified PCR results when a genetic
  modification is present in a product.

Figure 5. A subset of genomic DNA extraction
samples is shown in this gel. Lane 1 l-Hind
III/fX174 Hae III DNA ladder. Lane 2 Corn
muffin. Lane 3 Corn tortilla. Lane 4 Soft
tofu. Lane 5 Silken tofu. Lane 6 Roundup
Ready soya seeds. Lane 7 Roundup Ready corn
seeds. DNA was successfully extracted in all
above samples except for corn tortilla as
indicated for the lack of stain with ethidium
bromide in the polaroid picture.
Figure 6. A subset of PCR corn samples is shown
in this gel. Read these samples results in pair
lanes (3 and 4 5 and 6 7 and 8). Lane 1 100
base pairs ladder. Lane 2 No DNA, negative
control. Lane 3 Moss corn meal specie specific
PCR reaction. Lane 4 Moss corn meal genetic
modification PCR reaction. Lane 5 White Lilly
corn bread mix specie specific PCR reaction.
Lane 6 White Lilly corn bread mix genetic
modification PCR reaction. Lane 7 Jiffy corn
muffin specie specific PCR reaction. Lane 8
Jiffy corn muffin genetic modification PCR
reaction. PCR product for maize invertase specie
specific DNA fragment is 226 bp while the genetic
modification of CRY1Ab PCR product is 184 bp as
indicated by the stain with ethidium bromide in
the polaroid picture. The gel illustrates that
Moss corn meal and Jiffy corn muffin samples do
contain the CRY1Ab genetic modification as seen
in lanes 4 and 8, respectively when compared to
their controls lane 3 and 7, respectively.
Figure 7. A subset of PCR soy samples is shown
in this gel. Read these samples results pair
lanes (3 and 4 5 and 6 7 and 8) Lane 1 100
base pairs ladder. Lane 2 No DNA, negative
control. Lane 3 Roundup-ready soy seeds specie
specific PCR reaction. Lane 4 Roundup-ready soy
seeds genetic modification PCR reaction. Lane 5
Friedas soft tofu mix specie specific PCR
reaction. Lane 6 Friedas soft tofu mix genetic
modification PCR reaction. Lane 7 Mori-Nu
silken tofu specie specific PCR reaction. Lane
8 Mori-Nu silken tofu genetic modification PCR
reaction. PCR product for soy lectin specie
specific DNA fragment is 318 bp while the genetic
modification of CRY1Ab PCR product is 356 bp as
indicated by the stain with ethidium bromide in
the polaroid picture. The gel illustrates that
Roundup-ready soy seeds used as positive control
indeed contains the CP4-EPSPS genetic
modification as seen in lane 4 when compared to
its control lane 3.
Methods

• Purchase food products containing corn or soy
  from local grocery stores.
• Products were ground and weighed for genomic DNA
  extraction according to one of these
• methods Dneasy Plant Mini Kit from Qiagen ,
  Guanidine HCl, or Salt-extraction technique.
• Electrophoresis was used to check the integrity
  of the genomic DNA extracted.
• DNA amplification was performed by Polymerase
  Chain Reaction (PCR) (figure 4). Two
• different reactions were performed to detect
  specie specific organisms and to detect the
  specific
• genetic modification.
• 1. In corn samples, the primers used
  target the maize invertase gene while in soy
  samples,
• the primers used target the soy
  lectin gene.
• 2. The genetic modification for corn
  samples is CRY1Ab and, for soy is CP4-EPSPS.
• Electrophoresis was used to check the size of the
  PCR products.

Table 1
Figure 1. Ras-Mol CRY protein structure. The
different colors in the protein chain indicate
its secondary structure red is a-helix, mustard
is b-sheet and blue/grey is a turn or loop.
Water molecules surrounding the protein are shown
in yellow. The domain identity is shown with
roman numerals. (PDB 1DLC)
Future work
Corn Products Assessed for CRY1Ab Modification
Table 2
Figure 4.

• Improve DNA extraction by using other
  procedures so that all products collected could
  be assessed for genetic modifications.
• Analyze more samples to collect data for the
  consumer data bank.
• Compare the PCR results to other PCR reactions
  in which the target DNA is the Cauliflower mosaic
  virus promoter or the Glycine max transgenic
  Agrobacterium tumafaciens nos gene-terminator
  region.
• Quantify all PCR results.

Soya Products Assessed for CP4-EPSPS Modification
CP4-EPSPS is the abbreviation for the enzyme
CP4-5-Enolpyruvylshikimate-3 Phosphate Synthase
and it is naturally found in the common soil
microorganism Agrobacterium tumefaciens sp.
strain CP4. Since the active ingredient in
Roundup Ready Herbicides is glyphosate, this
molecule is the target of the genetic
modification. While native EPSPS wild-type
protein in soybeans is sensitive to glyphosate,
the CP4-EPSPS Roundup Ready herbicide protein
confers resistance to glyphosate. Glyphosate
interacts with the EPSPS enzyme (Figure 2),
inhibiting the catalysis of an essential step in
the aromatic amino acid synthesis pathway in
plants, many bacteria, and microbes (Figure 3).
References
1. Aljanabi C. and Martinez I. 1997. Universal
and rapid salt extraction of high quality genomic
DNA for PCR-based techniques. Nucleic Acids
Research. 2522. 4692-4693. 2. Bernstein J.A.,
Bernstein I.L., Bucchini L., Goldman L.R.,
Hamilton R.G., Lehner S., Rubin C. and Sampson
H.A. 2003. Clinical and Laboratory
Investigation of Allergy to Genetically Modified
Foods. Environmental Health Perspectives 1118.
1114-1121. 3. Safety Assessment of YieldGardR
insect-protected corn event MON 810. 2002.
1-28. 4. Schonbrunn E., Eschenburg S.,
Shuttleworth W.A., Schloss J.V., Amrhein N.,
Evans J.N.S., and Kabsch W. 2001. Interaction
of the herbicide glyphosate with its target
enzyme 5-enolpyruvylshikimate 3-phosphate
synthase in atomic detail. PNAS. 984.
1376-1380. 5. Tengel C., Schubler P., Setzke E.,
Balles J., and Sprenger-Haubels M. 2001.
PCR-Based Detection of Genetically Modified
Soybean and Maize in Raw and High Processed
Foodstuffs. Biotechniques. 31 2. 426-429.
Results

• DNA extraction of 35 samples have been
  attempted, 19 extraction procedures were
• successful.
• Of these, 13 corn DNA samples were analyzed
  and 2 show the presence of CRY1Ab
• genetic modification (Table 1). While 6 soy
  DNA samples were analyzed and 1 shows
• the presence of CP4-EPSPS genetic
  modification (Table 2).
• There is no indication of the use of genetic
  modified organism on the products labels of those
• samples where genetic modifications were
  detected.
• This data bank may serve as a resource for
  consumer education and future reference
• should food allergies be reported.

Figure 2. Ras-Mol EPSPS enzyme structure. In
this figure, EPSPS shows its interaction with
Shikimate 3-Phosphate (white) and Glyphosate
(green). The different colors in the protein
chain indicate its secondary structure red is
a-helix, mustard is b-sheet and blue/white is a
turn or loop. The blue molecules are formic
acid. (PDB 1G6S)
Acknowledgments
This work was presented as a talk at the 82th
Virginia Academy Science (May 27, 2004) and it
was financed by the Longwood University Student
Fund for Research. We want to thank Professors A.
Fink, M. Fink, E. Kinman, G. Lutz, and M. Zwick
for very helpful comments on the preparation of
the talk as well as of this poster.