In the Laboratory
Testing for Genetically Modified Foods Using PCR
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Ann Taylor* Department of Chemistry, Wabash College, Crawfordsville, IN 47933; *
[email protected] Samin Sajan Department of Biology and Biomedical Sciences, Washington University in St. Louis, St. Louis, MO 63130
The presence of genetic modification in foodstuffs has caused considerable concern, especially in European Union countries. A nice review of genetically modified foods was published in this Journal (1). As more countries require that genetically modified foods be labeled, a quick, easy, and accurate test for the presence of genetic modifications is necessary. The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA. It is commonly used to test for the presence of genetic modifications (2). In this laboratory exercise, students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S promoter. Similar experiments have been designed to analyze phage DNA (3), forensic samples (4), and known transgenic plant material (5); however, this is the first example that examines actual food products available at the grocery store. Background of Genetic Modification The biggest challenge facing farmers is controlling weeds and diseases while maintaining crop yield and quality. It is important that these objectives are met cheaply, as the average acre of corn earned $295 per year over the last ten years (6). That quantity of money needs to pay for the cost of seed, equipment, fertilizer, weed control, land, and the labor of the farmers (i.e., salary for the year). The most commonly used methods for achieving good weed control, disease management, crop yield, and quality are mass-methods such as pesticides and herbicides. A new approach for addressing these issues is through the production of genetically modified plants. In fact, 75% of soybeans and 34% of corn planted in 2002 were genetically modified (7). However, there are significant concerns about the environmental impact and possible allergenicity of genetically modified crops. There are two common types of genetically modified crops: insect resistant (Bt) and herbicide resistant (Roundup Ready). “Bt” is short for Bacillus thuringiensis, a soil bacterium whose spores contain a protein that is broken down in an insect’s gut to release a toxin, known as a delta-endotoxin. This toxin binds to and creates pores in the intestinal lining, resulting in an ion imbalance, paralysis, and after a few days, death of the insect (8). The most common herbicide resistance products are the “Roundup Ready” (RR) plants produced by Monsanto. Roundup is the commercial name for glyphosate, an inhibitor of an enzyme required for aromatic amino acid synthesis in plants. RR plants contain the bacterial version of this enzyme, which is not affected by glyphosate. To make a genetically modified plant, several components are needed in addition to the gene for the protein of interest. First, a method for getting the gene into the plant is www.JCE.DivCHED.org
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needed. Most commonly a plasmid from Agrobacterium tumefaciens called Ti is used. Alternatively, a “gene gun” can be used. Once the gene is in the plant, it must be correctly transcribed and translated. This requires a promoter sequence and species-specific codon usage. Usually a powerful promoter from cauliflower mosaic virus (CaMV 35S) is used. Experimental Overview This experiment evaluates commercially-available cornmeal products for the presence of the CaMV 35S promoter by PCR. Each PCR cycle consists of three steps: (i) heat denaturation of the double-stranded DNA, (ii) anneal sequencespecific primers onto the single-stranded DNA by cooling, and (iii) elongation of the primers to form a new DNA strand by a heat-resistant Taq polymerase. This cycle is repeated 30 to 40 times. DNA sequences located between the primers are ideally amplified 2n−2 times, where n is the number of times the cycle is repeated. This amplified DNA sequence can be detected by electrophoresis in an agarose gel. The experiment familiarizes students with DNA isolation and PCR as well as current issues in biochemistry. It is appropriate for a biochemistry course or an analytical chemistry course that integrates bioanalytical methods. The materials required for DNA isolation and PCR amplification are readily available as kits from standard biochemical suppliers. The thermocycler required for PCR may either be found in a biology department or can be built for $25 (9). To further enhance students’ understanding of the issues surrounding genetically modified foods, students can complete a case study that explores the concerns about genetically modified foods (10). The case study nicely complements the laboratory as it provides students with a productive activity during the incubation and electrophoresis steps of the procedure, thus avoiding the “hurry up and wait” syndrome of biochemistry laboratory activities. Experimental Procedure This laboratory experiment and case study is conducted over a two-week period in a one-semester biochemistry course. The students in the class are junior and senior chemistry and biology majors. Students work in pairs and choose four samples to evaluate, including Roundup Ready corn seed, nontransgenic corn seeds, and a variety of corn meals and corn-muffin mixes. During the first week, students isolate DNA from the corn-meal samples. Corn samples are digested with Proteinase K, then DNA is affinity-purified using Wizard columns from Promega (11). The use of this purification system familiarizes students with a commonly used commercial kit, allows the use of small sample sizes, and preparation of multiple samples by each pair of students, and
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Journal of Chemical Education
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In the Laboratory
fin mixes did not show the presence of the CaMV promoter. Throughout the procedure, it is important that students avoid cross contamination between samples (i.e., use separate pipette tips when loading different samples). Comments from students who performed this laboratory were very positive. They appreciated the relevance to real life, and when it was partnered to the case study, students cited this lab as enjoyable and informative. This activity could easily be adapted for a bioanalytical class to address the issues of sensitivity and selectivity, which are crucial in using PCR to test food products (12). The effect of varying PCR parameters, especially cycle number, and the use of an internal standard would reveal some of the technical issues that are involved in designing a standard protocol for testing samples of interest (13). Figure 1. Agarose gel of the PCR products reveals the presence of amplified CaMV 35S promoter in corn samples. Lane 1, Arrowhead Mills corn meal; lane 2, Kroger corn meal; lane 3, Quaker white corn meal; lane 4, Cotton Pickin’ corn-muffin mix; lane 5, Jiffy corn-muffin mix; lane 6, Gold Medal corn-muffin mix; lane 7, Aunt Jemima corn-bread mix; lane 8, Martha White corn-muffin mix; lane 9, nontransgenic corn seed; lane 10, Pioneer YieldGard corn seed.
Acknowledgments This work was supported by Wabash College through the Haines Fund for the Study of Biochemistry. Pioneer Seed Company provided YieldGard seeds. W
avoids more hazardous DNA isolation systems, such as phenol兾chloroform extraction. Other methods of isolating DNA may be used, as long as sufficient quantity and purity of DNA is obtained. During the Proteinase K incubation time, small groups discuss their assigned topic from the case study. The quantity of DNA obtained and its purity are determined by the absorbance at 260 nm and the ratio of the absorbances at 260 nm and 280 nm. Between the class periods, PCR is conducted using primers for cauliflower mosaic virus 35S promoter, a common promoter used in the production of transgenic plants. During the second class period, the amplified samples are analyzed by agarose gel electrophoresis. While the gels are running, the small groups present the case studies. Hazards Ethidium bromide is a potent mutagen and should be handled with care. Gloves, goggles, and lab coat should be worn whenever working with solutions or gels containing ethidium bromide. Gels containing ethidium bromide should be disposed appropriately. UV eye protection must be used with transluminators that do not include built in safety shields. Results We have found that the 192 bp segment of the CaMV 35S promoter found in transgenic plants is easily amplified from corn meal and corn-muffin mixes. As shown in Figure 1, students successfully amplified the 35S promoter from Pioneer YieldGard seeds, (a positive control) as well as Kroger corn meal and Jiffy, Gold Medal, and Aunt Jemima brands of corn-muffin or bread mixes. Nontransgenic corn seeds, Arrowhead Mills (an “organic” product) and Quaker White corn meal, and Cotton Pickin’ and Martha White corn-muf598
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Supplemental Material
A student handout including background information as well as detailed instructions for the instructor are available in this issue of JCE Online. Also included is a case study on genetically modified food products, entitled The Case of the Shady Shells. Information for a general and an advanced biochemistry version with teaching notes and answer keys is available. Literature Cited 1. Popping, Bert. J. Chem. Educ. 2001, 78, 752–756. 2. Matsuoka, Takeshi; Kuribara, Hideo; Takubo, Ken; Akiyama, Hiroshi; Miura, Hirohito; Goda, Yukihir; Kusakabe, Yuko; Isshiki, Kenji; Toyoda, Masatake; Hino, Akihiro. J. Agric. Food Chem. 2002, 50, 2100–2109. 3. Weller, David L. J. Chem. Educ. 1994, 71, 340–341. 4. Millard, Julie T.; Pilon, André M. J. Chem. Educ. 2003, 80, 444–445. 5. Thion, L.; Vossen, C.; Couderc, B.; Erard, M.; Clemencon, B. Biochem. Molec. Biol. Educ. 2002, 30, 51–55. 6. USDA National Agricultural Statistics Service. http:// www.usda.gov/nass/pubs/trackrec/track03a.htm (accessed Dec 2004). 7. National Agricultural Statistics Service. http:// usda.mannlib.cornell.edu/reports/nassr/field/pcp-bba/acrg0602.txt (accessed Dec 2004). 8. Colorado State University. http://www.colostate.edu/programs/ lifesciences/TransgenicCrops/ (accessed Dec 2004). 9. Betsch, David F.; Blais, Brian S. Biochem. Molec. Biol. Educ. 2003, 31, 113–114. 10. Taylor, Ann T. S. J. Coll. Sci. Teach. 2004, 34, 46–49. http:// ublib.buffalo.edu/libraries/projects/cases/ubcase.htm (accessed Dec 2004). 11. Spoth, B.; Strauss, E. Promega Notes 1999, 73, 23–25. 12. Holst-Jensen, A.; Ronning, S. B.; Lovseth, A.; Berdal, K. G. Anal. Bioanal. Chem. 2003, 375, 985–993. 13. Bertheau, Y.; Diolez, A.; Kobilinsky, A.; Magin, K. J AOAC Int. 2002, 85, 801–808.
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