Download Detection of Genetically Altered Corn and Soybean Food Products

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 1990’s 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.
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.
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Research Question
Do any locally available foods contain CRY1Ab and/or CP4-EPSPS genetic modifications?
7 8
1000 bp→
1 2
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5 6 7
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500 bp→
4.4 Kbp→
500 bp→
300 bp→
2.3 Kbp→
200 bp→
200 bp→
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.
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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.
0.6 Kbp→
100 bp→
100 bp→
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.
Table 1
GMO
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 CP4EPSPS genetic modification as seen in lane 4 when compared to its control
lane 3.
No GMO
Future work
Corn Products Assessed for CRY1Ab Modification
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Figure 4.
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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).
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Results
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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)
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1.35 Kbp→
Methods
Figure 2.
5
1000 bp→
Figure 5.
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)
4
300 bp→
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Figure 1.
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Pyruvate (PEP) and Shikimate3-Phosphate (S3P).
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 insect’s 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.
III
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Figure 3. Chemical synthesis of 5-Enolpyruvylshikimate-3 Phosphate (EPSP) from Phosphoenol
23 Kbp→
II
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• 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.
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Jiffy corn muffin mix (1)
Moss’ plain yellow corn meal (1)
White Lily cornbread
Martha White cotton pickin‘ cornbread
Martha White buttermilk cornbread
Medallion tortilla chips-white corn
Del Monte canned corn
Great Value yellow corn muffin mix
Hungry Jack pancake mix
Roundup Ready corn seeds
Great Value taco shells
Gerber baby food: sweet potatoes/corn
La Banderita tortilla
Kellog’s frosted flakes cereal
Kellog’s corn flakes cereal
Argo corn starch
Del Monte creamy corn
FritoLay fritos corn chips
Wise cheez doodles
Nature’s Own wheat ‘n soy bread
General Mills Trix fruit corn puffs cereal
General Mills Kix crispy corn puffs cereal
Nature Valley Granola bar: corn meal
Wal-Mart fresh corn on the cob (May-2004)
General Mills apple cinnamon cheerios
V8 Splash smoothie strawberry-banana drink
Indian Head stone ground white corn meal
Moss’ plain white corn meal
Medallion nacho cheese tortilla chips
Clover brand cheerios
General Mills honey nut cheerios
General Mills cheerios
General Mills golden grahams
Martha White chocolate chip muffin mix
Martha White banana nut muffin mix
Jiffy corn muffin mix (2)
Moss’ plain yellow corn meal (2)
Winn Dixie crunchy cheese curls
Table 2
GMO
No GMO
Soya Products Assessed for CP4-EPSPS Modification
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Roundup Ready soy seeds
Friedas’ soft tofu
Soy max drink powder
Mori-Nu silken tofu
White Lily corn bread
Martha White cotton Pickin’ cornbread
Medallion tortilla chips-white corn
Great Value yellow corn muffin mix
Hungry Jack pancake mix
Roundup Ready corn seeds
Galaxy Foods veggie cheese slices
Slimfast oatmeal raisin bar
Plain soy milk
Slimfast success bar
Ham glaze
Kellogg’s frosted flakes cereal
Kellogg’s corn flakes cereal
Chocolate powder
Naturally Preferred vanilla soy milk
Wise cheez doodles
Nature’s Own wheat ‘n soy bread
Nature Valley Granola bar: corn meal
General Mills apple cinnamon cheerios
V8 Splash smoothie strawberry-banana drink
Medallion nacho cheese tortilla chips
General Mills golden grahams
Martha White chocolate chip muffin mix
Martha White banana nut muffin mix
Winn Dixie crunchy cheese curls
V8 Splash smoothie wild berry creme drink
V8 Splash smoothie tropical colada drink
• 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.
References
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Aljanabi C. and Martinez I. 1997. Universal and rapid salt extraction of high quality genomic
DNA for PCR-based techniques. Nucleic Acids Research. 25:22. 4692-4693.
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 111:8. 1114-1121.
Safety Assessment of YieldGardR insect-protected corn event MON 810. 2002. 1-28.
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 5enolpyruvylshikimate 3-phosphate synthase in atomic detail. PNAS. 98:4. 1376-1380.
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.
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.