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

1. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 1000 bp→ 1000 bp→ 23 Kbp→ 500 bp→ 4.4 Kbp→ 500 bp→ 300 bp→ 2.3 Kbp→ 300 bp→ 200 bp→ 1.35 Kbp→ 200 bp→ 100 bp→ 0.6 Kbp→ 100 bp→ I II III GMO • 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 • 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 No GMO No GMO GMO Detection of Genetically Altered Corn and Soybean Food Products by Polymerase Chain Reaction 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. 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 (Sesamiacretica) 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. 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-Phosphate (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 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 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 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. Consuelo J. Alvarez, Adrienne R. Hampton and Sara K. Lee Department of Natural Sciences, Longwood UniversityFarmville, VA 23909 USA 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 Agrobacteriumtumefaciens 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. 25:22. 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 111:8. 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. 98:4. 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.