GMO Food Project

Emi LeonardSchool of Chemistry and BiochemistryGeorgia Institute of Technology

November 22, 2010

Experimentation Dates: November 3 and November 10, 2010Date Due: November 22, 2010Date Submitted: November 22, 2010

All work provided here was the authors original work, and was done in compliance with the Honor Code for the Georgia Institute of Technology.

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INTRODUCTIONGenetic engineering of food involves the genetic modification of organisms. Many foods today are genetically modified or contain genetically modified ingredients. Americans have invested billions of dollars developing bigger, healthier, better-tasting, pest-resistant, disease-resistant crops, including tomatoes the food focused on in this experiment [1]. However, despite the agricultural benefits of genetic modification, controversy exists about the possible adverse effects of genetic modification. The conflicts between organic farmers and environmentalists and research scientists and private industries remain in constant debate [2]. With the push for an organic, natural diet in America more recently, many farmers have resorted back to growing crops under controlled breeding. Selection of the most profitable, best-looking, and best-tasting foods to breed during the next growing season helps to reduce the need for genetic modification of plants through unnatural selection. Even with the recent focus on controlled breeding, a method for detecting genetic modification in food is necessary, as foods containing genetic modification should be labeled accordingly in grocery stores to keep consumers informed about their purchases.The goal of this experiment was to determine if grape tomatoes contain genetic modification. This was accomplished by amplifying detectable plant and GMO genes through polymerase chain reaction (PCR) and running the PCR products on an agarose gel to determine the fragment sizes of the bands for each sample. The specific genes detected by PCR were the Photosystem II chloroplast gene, the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator. The latter two are common vectors for genetic modification and are therefore utilized to test the samples for genetic modification. Tomatoes were the first genetically modified fresh fruits or vegetables commercially available (Flavr Savr tomatoes) [3]. Since then, GM tomatoes have disappeared from the market. However, new developments in research on GM tomatoes have revived interest in them. New techniques would allow researchers to insert foreign DNA into the chloroplast rather than the nuclear DNA which would reduce concerns of genetic pollution via cross pollination. Other researchers have developed the worlds first salt-tolerant tomato [3]. While GM tomatoes havent yet returned to the market, the interest in them is increasing. The grape tomatoes utilized in this experiment were purchased from Publix and were from Santa Sweets, Inc. Santa Sweets, Inc. produces its grape tomatoes in Plant City, FL, Cedarville, NJ, and Nogales, AZ. Santa variety tomatoes are a first generation hybrid that are guaranteed to have not been genetically altered by the company [4]. Therefore the hypothesis for this experiment was that grape tomatoes are not genetically modified.

EXPERIMENTAL PROCEDURESMaterials. The protocol and materials were adapted from the GMO Investigator Kit produced by Bio-Rad. United States Santa Sweets, Inc. grape tomatoes were purchased from the Publix supermarket in Atlantic Station in Atlanta, GA and kept refrigerated for one day prior to use. Bio-Rad certified non-GMO grain and GMO DNA solution were used as the negative and positive control, respectively. The Bio-Rad InstaGene matrix was used for DNA extraction. PCR plant and GMO primers detecting for the Photosystem II chloroplast gene and the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator genes, respectively, were purchased from Bio-Rad. PCR was run on the Bio-Rad MyCycler thermal cycler. The molecular weight marker and Orange G loading dye obtained from Bio-Rad, and the GelStar obtained from Lonza were provided for electrophoresis. The PCR products were run on an agarose gel in an Owl EasyCast MiniGel System (Thermo Scientific). The gel was photographed using a Fotophoresis UV Transilluminator and a digital camera.DNA Extraction. Non-GMO grain, soaked for three hours in deionized water, and a sliced grape tomato (including skin, seeds and fruit) were homogenized into a pipettable slurry separately in deionized water using a mortar and pestle. The plant control slurry and the grape tomato slurry were added to separate 500 L aliquots of InstaGene matrix. The plant control and food samples in InstaGene matrix were heated in a 95C water bath for 5 minutes. Both samples were then centrifuged in a Fisher Scientific Marathon microA centrifuge for 5 minutes at 5250xg. The resulting supernatant contained the DNA sample.Polymerase Chain Reaction. Six PCR samples were prepared by adding Plant and GMO master mixes separately to the plant control, GMO DNA solution, and grape tomato food sample. The Plant and GMO master mixes each contained PCR master mix (Taq DNA Polymerase, dNTPs, MgCl2, and buffer) and plant primers or GMO primers, respectively. PCR was run on the 6 samples according to the program described below in Table 1.

Table 1. PCR OverviewStepFunctionTemperature (C)Duration (min)Number of Cycles

Initial DenaturationDenature dsDNA9421

AmplificationDenature dsDNA94140

Anneal Primers591

Polymerize722

Final ExtensionPolymerize72101

HoldHold Temp41Indefinitely1

Agarose Gel Electrophoresis. A 2 % agarose gel was prepared using 1.0103 g of agarose and 50 mL of 1X TAE buffer. GelStar (1L) was added to the warm agarose prior to pouring; the gel was allowed to solidify. Orange G loading dye was added to each PCR product and the PCR molecular weight marker in a 1:5 dye to sample ratio. The 6 samples and marker were loaded into the gel, and the gel was electrophoresed at 120 V for 1 hour. The gel was photographed using a Fotophoresis UV Transilluminator and a digital camera.

RESULTSThe agarose gel (Figure 1) of the PCR products revealed the DNA amplification of plant and GMO genes. The PCR molecular weight marker separated into bands of known sizes: 1000, 700, 500, 200 and 100 base pairs. The migration distances of these bands were measured in pixels using the Paint function of Microsoft. Estimated fragment sizes for the bands in the sample lanes were determined (Table 2) from the linear regression of the graph of logMW vs. migration distances of the molecular weight marker bands (Figure 2).Lanes 3, 4, and 5 represent the results for the GMO master mix samples. Lane 3 was loaded with non-GMO plant DNA and GMO master mix. No significant bands (GMO DNA-containing) appeared in this lane, as expected. Lane 4, containing GMO DNA (positive control) and GMO master mix, showed a bright band at a migration distance corresponding to a fragment size of 157 base pairs. This fragment size is slightly less than the expected 203 bp or 225 bp for the GMO DNA, but still suggests a successful positive control. Lane 5 containing the grape tomato DNA and GMO master mix displayed no significant bands. Lanes 3, 4, and 5 also showed bright bands at a fragment size of 74 or 79 base pairs, suggesting the presence of primer dimers in all three lanes. Lanes 7, 8, and 9 represent the results for the plant master mix. Lane 7, loaded with non-GMO plant DNA (negative control) and plant master mix, showed a band at 420 base pairs and at 87 base pairs. Lane 8 was loaded with GMO DNA and plant master mix and displayed a band at 372 base pairs. Lane 9, containing grape tomato DNA and plant master mix, displayed bands at 459 base pairs and 90 base pairs. The known fragment size of the Photosystem II chloroplast gene amplified in the plant master mix was 455 base pairs; fragment sizes of 420, 372 and 459 base pairs correspond to this gene. This suggests that the negative control was also successful. The bands in lanes 7 and 9 at fragment sizes of 87 and 90 base pairs represent primer dimers. Lane 8, however, lacks primer dimers.The gel had some smearing in the lanes (lanes 4 and 8) containing GMO DNA; these lanes also displayed bands with smaller fragment sizes than expected. The bands appeared broader and less defined than expected in all lanes, suggesting an error in the overall electrophoresis. However, in general bands appeared where expected and most had minimal smearing illustrating the success of the DNA extraction and PCR.

Figure 1. Agarose gel (2%) of PCR molecular weight marker and 6 samples. Lanes 3-5 represent samples with GMO master mix and Lanes 7-9 represent samples with plant master mix. P stands for the plant sample (negative control), G stands for the GMO sample (positive control), and F stands for the food sample (grape tomatoes).

Figure 2. Plot of Standard Curve of the PCR Molecular Weight Marker. By measuring the migration distance of the bands in the MW marker lane, a linear regression function of the logMW versus the migration distance (in pixels) graph was calculated. This function was used to determine the fragment sizes corresponding to the bands in each of the sample lanes.Table 2. Fragment Sizes of the Bands in the Agarose Gel Calculated from the Standard CurveMigration Distance (pixels)Molecular Weight (base pairs)

GMO Master MixPlant160474

GMO1332157

158079

Grape Tomatoes158079

Plant Master MixPlant976420

154487

GMO1020372

Grape Tomatoes944459

153290

DISCUSSIONThe goals of this experiment were to extract DNA from plant samples and to run an agarose gel of PCR products to test for genetic modification of a grape tomato. Polymerase chain reaction was used to amplify DNA fragments coding for detectable plant and GMO genes. The detectable plant gene was the Photosystem II chloroplast gene, and the detectable GMO genes were cauliflower mosaic virus 35S promoter and the nopaline synthase terminator genes. Since the plant and GMO primers only amplify DNA coding for the detected genes, only sample DNA containing the coding DNA sequence was amplified. Agarose gel electrophoresis, used to separate DNA samples by fragment size, was run on the PCR products to check for the presence of bands corresponding to fragment sizes of the amplified DNA sequences of the plant and GMO genes.Overall Conclusions. The results of the agarose gel revealed that Santa Sweets, Inc. grape tomatoes are not genetically modified. The extracted DNA from the grape tomato test sample did not contain the DNA sequence encoding for the cauliflower mosaic virus 35S promoter or the nopaline synthase terminator genes, demonstrating that the grape tomato tested had not been genetically modified. Bands appeared at 420, 372 and 459 base pairs for the plant, GMO and grape tomato samples, respectively, when combined with plant master mix. Since the expected size of the Photosystem II chloroplast gene is 455 base pairs, these bands verify that these samples are all plants, or more precisely, photosynthetic organisms. The grape tomato sample contains a band at 459 base pairs confirming that it is a plant (and that DNA extraction was successful).For the samples prepared with GMO master mix, only the GMO positive control showed a band at 157 base pairs. The expected fragment size for the GMO detectable genes is 203 or 225 base pairs; therefore this band confirms the presence of genetically modified DNA in the GMO positive control DNA. The presence of a band at 420 bp for the plant sample and a band at 157 bp for the GMO sample, as well as the lack of a band around 200 bp in the non-GMO plant sample demonstrate the success of the negative and positive controls, validating the conclusions made. The grape tomato sample lacks a band near 200 base pairs suggesting that it is not genetically modified. The success of both controls paired with the results support the conclusion that the grape tomato tested was not genetically modified by the detectable genes.Anomalies. The gel had some smearing in lanes 4 and 8 containing GMO DNA; these lanes also displayed bands with smaller fragment sizes than expected. This was most likely due to an excess of DNA in these samples. The bands in these lanes were extremely wide and intense suggesting a great deal of DNA was present; an excess of DNA would result in smearing and would force the sample to spread out, skewing the calculated fragment size as seen. The bands also appeared broader and less defined than expected in all lanes, suggesting an error in the overall electrophoresis. This was probably due to an excess of DNA in all samples (too much was loaded into each well) and to stopping the electrophoresis too early. As discussed earlier, excess DNA results in wider bands. Also, the longer a gel runs, the further the fragments are allowed to migrate creating the separation desired in a gel. Had the gel been run for as little as ten more minutes, it is possible that the fragments would have separated further creating tighter, more defined bands. Five of the six samples displayed bands between 74 and 90 base pairs, corresponding to primer dimers. Primer dimers occur when two primers anneal due to complementarity in their 3 ends. During polymerization they are extended resulting in primer dimers that are approximately twice the size of the initial primer. During the following the cycles, these primer dimers continue to compete for binding to the primer resulting in amplification and therefore visibility on the gel [5]. As primers tend to be 30-50 base pairs, the bands seen between 74 and 90 base pairs are about twice this and most likely correspond to primer dimers. This anomaly occurs in both the plant and GMO primers; however, the GMO primer dimer bands are much more intense than those of the plant primer dimers. This is possibly due to the sequence of the primers. Primer dimers form more readily if the initial annealing of the primers is stable. Constructs that contain more G-C pairs or overlap longer are more stable [5]. It is possible that the DNA sequences of the GMO primer dimers either overlap further or contain more G-C pairs than those of the plant primer dimers. It should also be noted that the GMO DNA in lanes 4 and 8 seems to contain fewer primer dimers than the surrounding lanes. Lane 8 actually contains no primer dimers. A possible explanation of this phenomenon is that the large amount of complementary DNA out-competes the primer for annealing. In lanes 3 and 5 the primers had no complementary DNA to bind to, so they seem to have simply bound to themselves resulting in the large quantity of primer dimers. In lane 4, however, the primers had a higher affinity for the GMO DNA than for themselves, resulting in the lower intensity of the primer dimer band. In lanes 7 and 9, the primers had a higher affinity for the plant DNA so the primer dimer bands are less intense than the plant bands. However, in lane 8 the huge quantity of GMO plant DNA resulted in complete primer-plant DNA annealing and no primer dimer formation. Broader Impact. The controversy with genetically modified organisms, primarily crops, raises health and environmental concerns. Researchers and scientists are constantly searching for a safe and effective way to enhance crops without creating unpleasant effects. The results of this experiment illustrate the effectiveness of controlled breeding of plants as a possible compromise. The grape tomato tested was ripe, fresh, plump, and yet not genetically modified despite the fact that it is currently not in season. It was instead grown under controlled breeding and progressive growing practices.As the genetic modification of crops becomes more and more popular, reliable methods for detecting genetic modification are necessary for traceability of these plants. Due to low concentrations of DNA and difficulties in extracting and isolating DNA from plants, detecting genetic modification in plants has proven more difficult than expected. This experiment proposed just one method for detecting genetic modification, but there are genes other than the CaMV 35S promoter and NOS terminator used to genetically modify plants that the method failed to detect. Also, with increasing technology in the field, detection of the genetic modification must remain cost-efficient. Development of microarrays, mass spectrometry, biosensors, and near infrared spectroscopy techniques are the main focus of possible new detection methods [6].Overall, DNA extraction and PCR were successful techniques applied to amplify DNA sequences coding for plant and GMO detectable genes. The agarose gel run on the PCR products revealed that the Santa Sweets, Inc. grape tomatoes were not genetically modified to contain either the CaMV 35S promoter or the NOS terminator genes. Therefore, the hypothesis, stating that the grape tomatoes were not genetically modified, was accepted by the results of this experiment.

REFERENCES[1] Ambra R; Azzini E; Durazzo A; Foddai MS; Maiani G. (2008) Assessment of the nutritional values of genetically modified wheat, corn and tomato crops, J Agric Food Chem 56, 9206-9214.

[2] Dona, Artemis; Arvantioyannis, Ioannis S. (2009) Health Risks of Genetically Modified Foods, Critical Reviews in Food Science and Nutrition 49, 164-175.

[3] Bukenya, J; Wright, N. (2007) Determinants of Consumer Attitudes and Purchase Intentions With Regard to Genetically Modified Tomatoes, Agribusiness 23, 117-130.

[4] www.santasweets.com (used to obtain product information)

[5] Chou, Q; Russell, M; Birch, D; Raymond, J; Bloch, W. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications, Nucleic Acids Research 20, 1717-1723.

[6] Lu, J; Shi, X; Mo, Q; Li, X. (2008) Safety problems and detection technology of genetically-modified foods, Xiandai Yufang Yixue 35, 3951-3953.2