Can voluntary non-GMO labels be trusted? Testing for the presence of the CaMV 35S promotor in soy-containing food products with polymerase chain reactions.

Can voluntary non-GMO labels be trusted? Testing for the presence of the CaMV 35S promoter in soy-containing food products with polymerase chain reactions.

Ingrid Schoonover

October 17, 2019

Cellular Biology

Abstract

Genetically modified organisms contain the DNA of a different species, this practice of genetic engineering is common in the agricultural industry for the purpose of increasing nutritional content, increasing crop yields, and creating herbicide or insecticide resistance. One of the most common ways agricultural crops are genetically modified is with the CaMV 35S promoter, which can have origins traced back to the Cauliflower Mosaic Virus that destroyed crucifer vegetables in the 1900s. Despite the benefits that genetic engineering can provide there are drawbacks as well; the most frightening is the increased risk of a severe allergic reaction that can come when a consumer eats a food product containing the DNA of another species that they are allergic to. The United States does not have any federal regulations in place for the regulation of GMO labels, this has led to a grassroots movement of voluntary non-GMO labeling amongst companies. This experiment will be tested two products, one labeled as non-GMO and the other unlabeled, for the presence of GMOs to determine the reliability of voluntary labeling. The hypothesis stated that Nasoya Organic Cubed Super Firm Tofu, which was labeled as non-GMO, would not be genetically modified; while the belVita breakfast biscuit blueberry flavor, which lacked any such labels would be genetically modified. It was predicted that when the 35S CaMV primer was used that the belVita breakfast biscuit would contain the CaMV 35S promoter, with a molecular size of 162 base pairs; and that the Nasoya Tofu would not contain the CaMV 35S promoter, so would not have any bands that were 162 base pairs in length. To achieve these results the DNA of both food items, a negative control (wildtype soy), and a positive control (Roundup-Ready soy) was isolated with Edward’s Buffer, isopropanol, and TE/RNase A buffer. Samples of isolated DNA were prepared for PCR with Taq polymerase and primed with either Tubulin or 35S CaMV. After PCR amplification the DNA samples were run through gel electrophoresis so that the DNA fragments could be observed under UV light and analyzed. As predicted the Nasoya Tofu did not contain the CaMV 35S promotor because it lacked DNA with a molecular size of 162 base pairs, confirming the first part of the hypothesis. However, the second part of the hypothesis was disproved because the belVita breakfast biscuit did not contain the CaMV 35S promoter as predicted because it lacked DNA with a molecular size of 162 base pairs. The absence of the CaMV 35S promoter led to the conclusion that neither of the products were genetically modified. Thus, it is generally true that products with labels verified by the non-GMO project are accurate.

Introduction

Genetically modified organisms have always stirred up a controversy, and this is no exception for genetically engineered crops. A genetically modified organism (GMO) is an organism that has had its genome genetically engineered by introducing the DNA of another species. The history of GMO crops goes back to the discovery of the Cauliflower Mosaic Virus (CaMV) which devasted crucifer vegetables in the mid-1900s (Somssich, 2018). Unlike single-stranded retroviruses, CaMV has double-stranded DNA that allows it to enter plant cells (Somssich, 2018). This virus was so successful because it could code its own genome into that of the plant; the 35s mRNA are involved in the process of DNA replication (Somssich, 2018). Scientists exploited this opportunity to genetically engineer plants and quickly isolated the DNA segment that could be used as a promotor for other genes. The promotor is the region of DNA that comes before a gene sequence and controls how that gene is expressed in the organism (Somssich, 2018).

Herbicides are commonly used in commercial agriculture to kill weeds, unfortunately, this also harms the crops. One of the most commonly used herbicides is glyphosate, which targets the EPSP synthase enzyme and kills the plant by depriving it of essential nutrients (Somssich, 2018). The first application of the CaMV 35S promoter was to increase the expression of a version of the EPSP synthase that would make plants resistant to the negative effects of glyphosate; thus, the herbicide-resistant crops were born (Somssich, 2018). The company Monsanto created Roundup Ready soybeans which are Roundup-resistant; Roundup is an herbicide that uses glyphosate as its main ingredient (Goldbas, 2014).

Many scientists argue that genetically engineering crops to have herbicide-resistance is environmentally beneficial because it can translate to fewer herbicide applications (Nodoushani, Sintay, and Stewart 2015). On the flip side, research has shown that Roundup-Ready soy contains herbicide residues, contains fewer nutrients than non-GMO soy, and can disrupt female hormones (Goldbas, 2014). Furthermore, GMO crops can cause severe or fatal allergic reactions (Nodoushani, Sintay, and Stewart 2015). In the United States companies do not have to disclose if their products are GMO (Bain and Dandachi, 2014), so the unsuspecting consumer with a soy allergy may have an allergic reaction if they eat corn that contained the DNA of a soy plant, this can be potentially problematic since 80% of processed foods and 60% of fresh vegetables (DiCicco-Skinner, 2019) contain GMOs (Bain and Dandachi, 2014). Many GMO advocates dispute these claims by countering them with the possible benefits, for example: fighting world hunger by increasing levels of minerals and vitamin A in GM crops (Goldbas, 2014), reduce food waste by increasing GM crop shelf life, and reducing resource waste by engineering crops to use less water while simultaneously increasing yields (Nodoushani, Sintay, and Stewart 2015).

While the United States federal government has failed to set up any regulations for labeling GMO-products this hasn’t stopped the emergence of independent GMO-labeling organizations, such as the Non-GMO Project which sets standards for voluntary-labeling of non-GMO foods, allowing up to 0.9% genetically engineered DNA in certified products (Bain and Dandachi, 2014). Without federal regulations in place to control the labeling of non-GMO products it is possible that companies are exploiting these labels to increase sales and appeal to health-conscious consumers.

Fortunately, it is relatively easy to test food for the presence of GMOs with PCR and gel electrophoresis because 80% of GMO crops use the CaMV 35S promotor (Somssich, 2018). Thus, if a food sample contains the CaMV 35S promotor it has been genetically modified. The first step is to isolate kill the RNA and isolate DNA with a mixture of buffer-containing solutions, such as Edward’s Buffer and TE/RNase A Buffer (DeCicco-Skinner, 2019). Next, specific genetic sequences can be detected by polymerase chain reaction (PCR) (DeCicco-Skinner, 2019). It can be very difficult to observe a specific DNA molecule in a small sample, especially if only a couple copies are present, therefore PCR is used to amplify a region of DNA by making many copies of this one region (Bartee and Shriner, 2016). This next step requires two things: Taq DNA polymerase which is responsible for copying the DNA fragment and primers which direct the Taq polymerase to the DNA region of interest (Bartee and Shriner, 2016). For example, if a 35S CaMV primer is used for PCR then the Taq polymerase will amplify the region of the plant’s genome that contains the 35S CaMV promoter. The same process applies when Tubulin is used as the primer; then Taq polymerase amplifies the section of DNA that encodes for Tubulin. The polymerase chain reaction is done in three steps within a thermal cycler: DNA denaturation at 94 degrees Celcius separates the DNA helix into two strands, DNA annealing at 60 degrees Celcius allows primers to bind to the complementary sequence within the plant genome, and DNA extension at 72 degrees Celcius builds a new DNA helix for the DNA region targeted by the primer (DeCicco-Skinner, 2019; Bartee and Shriner, 2016). The last step in the process of testing for GMOs involves gel electrophoresis, a chamber filled with agarose gel is given an electric field by positive and negative electrodes on either end (DeCicco-Skinner, 2019). DNA which is negatively charged is inserted into wells on the side of the chamber towards the negative electrodes, once the apparatus is powered the DNA will be attracted to the positive electrode on the opposite side of the chamber and thus move towards it (DeCicco-Skinner, 2019). DNA fragments with more base pairs will have a more difficult time moving through the gel in comparison to those will fewer base pairs in a given amount of time, thus the smaller fragments will move the greatest distance (DeCicco-Skinner, 2019). After gel electrophoresis is complete, the gel plate can be removed from the well and placed on a UV transilluminator for the purpose of observing the molecular sizes of the DNA that was amplified via PCR (DeCicco-Skinner, 2019).

This experiment will be testing two soy-containing products for the presence of GMOs by PCR and gel electrophoresis, GMOs would be indicated by the presence of the CaMV 35S promoter. The first food product, Nasoya Organic Cubed Super Firm Tofu, is part of the Non-GMO Project. The second product is belVita breakfast biscuit blueberry flavor, it has no labels indicating the presence or lack of GMOs. The goal with this is to test the reliability of voluntary non-GMO labels, additionally, this experiment will determine if a lack of GMO labels automatically indicates that the product has genetically engineered material. These two products, along with a negative control (wildtype soy) and a positive control (Roundup-Ready soy) will undergo DNA isolation, amplification by PCR using two different primers, and gel electrophoresis to test if they are GMO. The first primer is CaMV 35S, it has a molecular size of 162 BP so the positive control and any GMO samples should display a 162 BP band. The second primer is Tubulin, which essentially serves as another control to ensure the experiment is valid. Tubulin is a protein found in all organisms (DeCicco-Skinner, 2019) because it forms the microtubules that create the so-called train tracks of the cell which allow for movement within the cell (Yarris, 1998). Tubulin has a molecular size of 185 BP so all of the samples including the controls should display a 185 BP band.

It is hypothesized that the Nasoya Organic Cubed Super Firm Tofu will not contain the DNA of genetically modified soybean, because the packaging had a label that said it is NON-GMO project verified. Additionally, the belVita breakfast biscuit blueberry flavor will contain the DNA of genetically modified soybean. It is suspected that the belVita will contain GMO soybean because it contains soy lecithin and 94% of soybeans are GMO (Goldbas, 2014), and the packaging has no labels to otherwise indicate that it is non-GMO. Therefore, it is predicted that the gel electrophoresis of Nasoya Organic Cubed Super Firm Tofu using 35S CaMV as a primer will not produce any observable bands; but the gel electrophoresis of belVita breakfast biscuit blueberry flavor using 35S CaMV as the primer will produce observable bands at 162 BP. To ensure that the results of this experiment are valid there are three additional requirements that must be observed. First, the gel electrophoresis of the negative control containing wildtype or non-GMO soybean using 35S CaMV as a primer should not produce any observable bands. Second, the gel electrophoresis of the positive control containing Roundup-Ready or GMO soybean using 35S CaMV as a primer should produce observable bands at 162 BP. Third, all of the samples that undergo gel electrophoresis using Tubulin as the primer should produce observable bands at 185 BP.

Methods and Materials

Isolate DNA

A 1 cm2 piece of tissue was cut from the leaf of a Roundup ReadyTM (RR) soybean plant and placed into a 1.5 mL tube to serve as a positive control for this experiment. A similar amount of Nasoya Organic tofu and belVita blueberry flavor Breakfast Biscuits, both containing soybean, were crushed and placed in their respective tubes. Next, 100 μl of Edward’s buffer was added to each of the tubes, and a plastic pestle was used to grind up the contents. Then, 900 μl of Edward’s buffer was added to each of the tubes, and the contents were ground once more. The tubes were vortexed briefly and then heated at 65 degrees Celcius for 5 minutes on a heating block. These tubes were then spun in a microcentrifuge for 2 minutes and 350 μl of each supernatant was transferred to 3 new tubes containing 400 μl of isopropanol. The tubes were inverted to mix the liquids and then incubated at room temperature for 3 minutes. Next, the tubes were spun in a microcentrifuge for 5 minutes until nucleic acids formed a precipitate, at which point the remaining supernatant was pipetted out of each tube. The tubes were uncapped and placed in a flow hood for 10 minutes to speed the evaporation of the isopropanol. After the isopropanol had completely evaporated 100 μl of TE/RNase A buffer was added to each tube and mixed with the nucleic acid pellet. The tubes were incubated at room temperature for 5 minutes and then spun in a microcentrifuge for a minute so that the supernatant could be poured into new tubes (DeCicco-Skinner, 2019).

Amplify DNA by PCR

6 tubes of Ready-To-Go PCR Beads containing Taq polymerase, nucleotides, and salts were obtained. 22.5 μl of Tubulin promoter was added to three of the tubes and then 2.5 μl DNA from each of the three soybean tubes (control, Nasoya tofu, and belVita breakfast biscuit) was added to the 3 separate PCR tubes. Next, this process was repeated for the last 3 PCR but with 35S CaMV primer instead of Tubulin. The PCR tubes were vortexed and then spun with the PCR tube microcentrifuge before being placed on ice. Next, the PCR tubes went through a six-step thermal cycle: 1) 94 degrees Celcius for 2 minutes, 2) 94 degrees Celcius for 30 seconds, 3) 60 degrees Celcius for 30 seconds, 4) 72 degrees Celcius for 30 seconds (steps 2-4 were repeated 40 times), 5) 72 degrees Celcius for 7 minutes, 6) 4 degrees Celcius overnight. Then the samples were stored at -20 degrees Celcius for a week (DeCicco-Skinner, 2019).

Analyzing PCR Products by Gel Electrophoresis

A gel tray was gently placed into a casting rack, the walls were tightened to secure it in place. In a glass flask, 1.0 grams of agarose was combined with 50 ml of 1x TAE buffer and then heated in a microwave in 20-30 second intervals until the solution was clear, next 5 μl of 10,000x SYBR Safe DNA gel stain was added to the agarose mixture. After the mixture was poured into the gel tray a comb was added to the end of the tray to create wells, and then the casting rack and gel were placed in the fridge for 20 minutes so the gel could solidify. Next, the comb was removed from the gel, also the gel was transferred from the casting rack into the electrophoresis chamber with the wells towards the negative electrode and topped with 1x TAE buffer. This created 8 wells, the first well was filled with 25 μl of a pBR322/BstNI marker containing bands for the following molecular sizes: 121 BP, 383 BP, 929 BP, 1,058 BP, 1,857 BP. Next, 25 μl of each of the following PCR samples were loaded separately to the next 6 wells: Roundup Ready control with Tubulin in well #2, belVita breakfast biscuit with Tubulin in well #3, Nasoya tofu with Tubulin in well #4, Roundup Ready control with 35S CaMV in well #5, belVita breakfast biscuit with 35S CaMV in well #6, and finally Nasoya tofu with 35S CaMV in well #7. The electrophoresis chamber cover was attached by matching the red electrode to the red plug and the black electrode to the black plug. After this arrangement was verified the apparatus was run at 120V/90mA until the dye migrated halfway down the gel, which took about 45 minutes. After turning off the power supply and dissembling the apparatus, the gel was then moved to a UV transilluminator to be observed and analyzed (DeCicco-Skinner, 2019).

Results

Figure 1: Gel Electrophoresis results. Showing the bands from the pBR322/BstNI marker and molecular sizes of the samples that were amplified by PCR with either Tubulin or 35S CaMV as the primer. The negative control (wildtype soybean) was observed to have a band size of 185 BP when Tubulin was used as the primer and a band size of 162 BP when 35S CaMV was used as the primer (shown in Figure 1A). The positive control (Roundup Ready soybean) was observed to have a band size of 185 BP when Tubulin was used as the primer and a band size of 162 BP when 35S CaMV was used as the primer (shown in Figure 1B). BelVita breakfast biscuit was observed to have a band size of 185 BP when Tubulin was used as the primer and no bands when 35S CaMV was used as the primer (shown in Figure 1B). Nasoya Tofu was observed to have a band size of 185 BP when Tubulin was used as the primer and no bands when 35S CaMV was used as the primer (shown in Figure 1B).

Figure 1B displays the results from the experiment with belVita, Nasoya Tofu, and Roundup Ready soybean (positive control). Figure 1A displays the results from a different experiment but was included because it used wildtype soybean (negative control). Distinct bands were visible at the location where the pBR322/BstNI marker was inserted at the molecular sizes 1,857 BP, 1,058 BP, 929 BP, and 383 BP. Very faint but visible bands were seen at the molecular size 121 BP.

Tubulin was present at 185 BP for the wildtype soybean, roundup ready soybean, belVita, and Nasoya Tofu. When the samples were PCR amplified with the 35S CaMV primer a very distinct bold band appeared for the Roundup-Ready sample (positive control) at the 162 BP lane, and a very faint band appeared for the wildtype sample (negative control) at the 162 BP lane. No bands appeared for belVita or the Nasoya Tofu at 162 BP.
Additionally, incredibly faint bands were observed below the 121 BP line for the belVita and Nasoya Tofu primed with 35S CaMV.

Table 1: Expected and Observed results from Gel Electrophoresis of soybean-containing samples and controls.
Table 1 summarizes the expected versus observed molecular sizes of samples after gel electrophoresis. The pBR322/BstNI marker was expected and observed to display bands of the sizes: 1,857 BP, 1,058 BP, 929 BP, and 383 BP, and 121 BP.

The positive control (Roundup Ready) was expected to display a band size of 185 BP when Tubulin was used as the primer, and it was observed with a 185 BP band size. The belVita breakfast biscuit was expected to display a band size of 185 BP when Tubulin was used as the primer, and it was observed with a 185 BP band size. The Nasoya Organic Cubed Super Firm Tofu was expected to display a band size of 185 BP when Tubulin was used as the primer, and it was observed with a 185 BP band size. The negative control (wildtype soy) was expected to display a band size of 185 BP when Tubulin was used as the primer, and it was observed with a 185 BP band size.

The positive control (Roundup Ready) was expected to display a band size of 162 BP when 35S CaMV was used as the primer, and it was observed with a 162 BP band size. The belVita breakfast biscuit was expected to display a band size of 162 BP when 35S CaMV was used as the primer, yet, no bands were observed. The Nasoya Organic Cubed Super Firm Tofu was not expected to display any band sizes when 35S CaMV was used as a primer, and no band sizes were observed. The negative control (wildtype soy) was not expected to display any band sizes when 35S CaMV was used as a primer, yet, it displayed a band size of 162 BP.

Discussion

This experiment tested two soy-containing products for the presence of GMOs, which would be indicated by the presence of the CaMV 35S promoter, for the purpose of evaluating the accuracy of GMO labels. One of these products was Nasoya Organic Cubed Super Firm Tofu, it contained contains Water, Organic Whole Soybeans, Calcium Sulfate, Nigari (sea Water Extract). The packaging had a lot of labels for health-conscious consumers, one label said that it was organic, and another said that it was verified by the non-GMO Project. belVita breakfast biscuit blueberry flavor was the other products it contained a variety of ingredients including wheat, canola oil, riboflavin, natural flavor, soy lecithin, malt syrup from corn, and barley. Contrary to the tofu, it had no labels indicating the presence or lack of GMOs. Additionally, two controls were included in this experiment: a negative control containing leaves from wildtype (non-GMO) soybean plants and a positive control containing leaves from Roundup Ready (GMO) soybean plants. Including the controls was done for the purpose of having something to compare the food product samples to; meaning how the bands should appear if they do or do not contain genetically modified DNA.

The DNA of these two products, along with the negative control (wildtype soy) and the positive control (Roundup-Ready soy) was isolated and then amplified by PCR before undergoing gel electrophoresis to display molecular sizes of the different DNA fragments. Two different primers were used for the PCR amplification; CaMV 35S with a molecular size of 162 BP and Tubulin with a molecular size of 185 BP. A pBR322/BstNI marker was also included to display bands with the molecular sizes of 121 BP, 383 BP, 929 BP, 1,058 BP, and 1,857 BP.

The reason for using the CaMV 35S primer is because 80% of GMO crops use the CaMV 35S promotor (Somssich, 2018). The positive control, Roundup Ready soybean, is one of the GM crops that uses the CaMV 35S promoter, in this case, to engineer herbicide-resistance to glyphosate (Somssich, 2018). Therefore, the Roundup Ready soybean sample should display a band size at the 162 BP ladder, the other samples can be compared to this control to determine if they are also GMO; non-GMOs would not have a band at this location whereas GMOs would. The negative control, wildtype soybean, is not genetically modified so it should not have any bands at 162 BP.

All organisms contain a protein called Tubulin (DeCicco-Skinner, 2019), this is the protein that forms microtubules, part of a cell’s cytoskeleton (Yarris, 1998). All organisms need microtubules so that they can move organelles and vesicles within a cell (Yarris, 1998). Tubulin primer is not integral to the process of this experiment, but it increases the validity of results because the presence of a band at 185 BP for the samples ensures that the plant DNA was successfully isolated and amplified. If samples do not display the band at 185 BP, then it can be concluded that the DNA isolation was not successful, and the results would be declared invalid.

It was hypothesized that the Nasoya Organic Cubed Super Firm Tofu would not be genetically modified because a label on the packaging declared the item as part of a non-GMO project. On the other hand, it was hypothesized that the belVita breakfast biscuit blueberry flavor would be genetically modified because it contained soy lecithin yet did not indicate with any labels that the product is non-GMO. The reason for suspecting that is genetically modified is that 94% of soybeans are GMO (Goldbas, 2014), so statistically, it is likely to be GMO.

Therefore, it was predicted that there would not be any observable bands for Nasoya Organic Cubed Super Firm Tofu in the lane that used the 35S CaMV primer, because of the presence of bands at 162 BP in this lane would indicate the presence of genetically modified soybean. It follows then that there would be an observable band at 162 BP for belVita breakfast biscuit blueberry flavor in the lane that used the 35S CaMV primer. Additionally, it was predicted that there would be a band at 162 BP for the Roundup Ready soybean (positive control) but no bands for the wildtype soybean (negative control) in the lanes that used the 35S CaMV primer. Finally, all lanes that used the primer Tubulin should have observable bands at 185 BP because they should all living things should contain this protein.

Distinct bands were visible at the location where the pBR322/BstNI marker was inserted at the molecular sizes 1,857 BP, 1,058 BP, 929 BP, and 383 BP, and very faint but visible bands were seen at the molecular size 121 BP (Figure 1). This molecular ladder served as a point of comparison to determine the molecular size of other DNA that appeared in the results, such as the CaMV 35S promotor and Tubulin. The faintness of the 121 BP ladder can be explained by the length of run time; the electrophoresis was only allowed to run for 45 minutes so the small bands did not have an opportunity to travel as far, also it is faint simply because it is a smaller DNA fragment with only 121 base pairs as compared to 1,857 or 1,058 base pairs.

Additionally, two faint bands were observed in lanes 6 and 7 (Figure 1B). These are significantly below the 121 bands so are most likely 50 base pairs in length. This observation can be explained by the formation of primer-dimers. Recall that the experiment used primers in the PCR process (Tubulin and 35S CaMV), sometimes complementary nucleotide bonding occurs between these primers during PCR and forms a complex called primer-dimer, which is weakly held together by the intermolecular interaction (Brownie et al. 1997). They are more likely to form when higher concentrations of primer are present, or the PCR begins cold and increases in temperature during the cycles (Brownie et al. 1997). Conversely, primer-dimers are less likely to form when lower concentrations of primer are used, when high concentrations of Taq polymerase are used, or when the PCR begins at a very high temperature (Brownie et al. 1997).

Observations reveal that the samples contained only plant DNA because RNA would appear as a band 150 bp in length, and the samples did not have any bands of this length (Figure 1 and Table 1). The DNA isolation procedure involved three steps to ensure that RNA would not be present in the samples because the Taq polymerase used in the PCR process can only amplify. Edwards Buffer was added to the samples while they were ground, after putting them through a microcentrifuge the supernatant was removed from the samples and added to new tubes (DeCicco-Skinner, 2019). Isopropanol was added to these new tubes to encourage the DNA to precipitate into a pelletized form, the samples were spun in a microcentrifuge again and then all of the liquid was removed, and the samples dried in a flow hood (DeCicco-Skinner, 2019). To be sure that this pellet contained only DNA, a solution of TE/RNase A buffer was added to the tubes to dissolve the DNA and RNA, and then kill the RNA (DeCicco-Skinner, 2019). The supernatant that was removed from the samples after micro centrifuging again was the pure isolated DNA (DeCicco-Skinner, 2019).

The positive control (Roundup-Ready soybean) displayed a molecular band size of 162 BP when 35S CaMV was used as the primer (Figure 1B in lane 5 and Table 1), this was expected because it is genetically modified with the CaMV 35S promoter to have herbicide resistance. The Nasoya Organic Cubed Super Firm Tofu and the belVita breakfast biscuit with 35S CaMV primer were compared to this lane to determine if they also had the molecular band size of 162 BP. The Nasoya Tofu in lane 7 (Figure 1B) did not display a 162 BP band as predicted (Table 1). This supports the hypothesis that the Nasoya Tofu would not be genetically modified because as discussed earlier, the absence of the CaMV 35S promotor indicates that the crop is not genetically modified. The belVita breakfast biscuit in lane 6 (Figure 1B), also did not display a 162 BP band as predicted (Table 1). The absence of the CaMV 35S promotor disproves the hypothesis that the belVita breakfast biscuit would contain GMOs. However, there is more to examine before it can be concluded with certainty that these products do not contain GMOs because the three additional requirements that must be met for the hypothesis to be accepted.

First, the positive control (Roundup-Ready soy) should contain a 162 BP band when the 35S CaMV primer is used because this product has been genetically modified with the CaMV 35S promoter. The results show that this requirement was fulfilled because, in lane 5 (Figure 1B), the Roundup-Ready soybean had a 162 BP band. If this sample did not contain a band at 162 BP then it would mean that an error occurred, perhaps the wrong primer was added, or it could be the result of an issue with PCR or electrophoresis. It is necessary to use this control so the location of the molecular size can be visualized and compared to the unknown samples.

Second, the negative control (wildtype soy) should not contain a 162 BP band when the 35S CaMV primer is used because this product has not been genetically modified. The results show that this requirement was not fulfilled because in lane 5 (Figure 1A), the wildtype soybean had a faint 162 BP band. This led to the discovery that an error occurred during the production of the laboratory kit and GMO soybean had been sent instead of non-GMO soybean. This means, that this experiment does not have a negative control. This is slightly problematic because it brings the validity of the results into question because there is no guaranteed non-GMO sample to compare the food products to.

Third, all samples that used Tubulin as a primer should have bands at 185 BP because all of the samples came from living organisms and all organisms contain Tubulin to construct microtubules. The results show that all of the samples pertaining to this experiment had 185 BP bands when Tubulin was used as a primer (Table 1), meaning that the protein as present in all of the samples, this included: the wildtype soybean in lane 2 (Figure 1A), and the Roundup-Ready soybean, belVita breakfast biscuit, and Nasoya Tofu in lanes 2, 3, and 4 respectively (Figure 1B). This means that the procedure successfully isolated and amplified the DNA of these plant samples.

In summary, only two of the three requirements were achieved because human error meant that there was no negative control for this experiment. However, all of the other evidence, that being the comparison to the negative control, and presence of Tubulin, suggests that the experiment was successful so the results of the experiment can be defended as valid. That is to say, that both Nasoya Organic Cubed Super Firm Tofu and belVita breakfast biscuit blueberry flavor can be considered non-GMO because they do not contain the CaMV 35S promotor. The significance of this is that non-GMO labels, despite being voluntary, appear to be accurate and can generally be trusted.

It is surprising that the belVita breakfast biscuit is not genetically modified because it contains ingredients derived from soy and corn; the two most commonly genetically modified agricultural products in the United States. Literature shows that 90% of corn (Bain and Dandachi, 2014) and 94% of soy sold in the United States are genetically modified (Goldbas, 2014). It follows then that non-GMO products would want to distinguish themselves from the saturated market of GMO foods with non-GMO product labels to appeal to consumers who are part of the growing anti-GMO movement. However, research shows that this isn’t always the case, some companies strongly oppose labeling their products because it increases production costs, this is one possibility that could explain why the belVita was not labeled as a non-GMO product (Bain and Dandachi, 2014). An alternative explanation is that belVita is genetically modified but does not use the CaMV 35S promotor; while the majority of GM agricultural products use CaMV 35S, 20% of GM crops use a different promotor such as FMV 34S promotor (figwort mosaic virus) or the UBIQUITIN10 promotor (Somssich, 2018). This uncertainty can be reduced by improving the experiment to include other commonly used GM promotors as primers during PCR amplification. This means testing for the presence of CaMV 35S in the belVita and Tofu as well as for the presence of the FMV 34S promotor or UBQ1 promotor. Testing for a wide range of the promotors used in the genetically modified agricultural industry can increase the validity of concluding a product is non-GMO. Furthermore, this experiment should be repeated with an actual negative control, so that the food samples can be compared to an item that does not contain GMOs. Food plays such an integral role in human health for better or worse, therefore it is of the utmost importance that the prevalence of GMOs is known. This is particularly important for individuals with allergies, who may suffer from a serious life-threatening allergic reaction if they eat the food containing the DNA of a protein, they are allergic to.

References

Bain, C., and T. Dandachi (2014). Governing GMOs: The (Counter) Movement for Mandatory and Voluntary Non-GMO Labels. Sustainability 6: 9456-9476. doi:10.3390/su6129456.

Bartee, L., and W. Shriner (2016) Principles of Biology: Biology 211, 212, and 213: DNA Isolation, Gel Electrophoresis, and PCR. Open Oregon Educational Resources: Oregon State University.

Brownie, J., S. Shawcross, J. Theaker, D. Whitcombe, R. Ferrie, C. Newton, and S. Little (1997) The elimination of primer-dimer accumulation in PCR. Nucleic Acids Research 25(16): 3235-3241. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC146890/pdf/253235.pdf.

DeCicco-Skinner, K. 2019. Weeks 5-6: Detecting Genetically Modified Foods by PCR. Bio-320 Cell Biology lab American University: Washington, D.C. 6.

Goldbas, A. (2014) GMOs: What Are They? International Journal of Childbirth Education 29(3): 20-24. http://proxyau.wrlc.org/login?url=https://search-proquest-com.proxyau.wrlc.org/docview/1545045515?accountid=8285.

Nodoushani, O., J. Sintay, and C. Stewart (2015) Genetically Engineered Food and Genetically Modified Organisms. Competitive Forum 13(1): 136-141. http://proxyau.wrlc.org/login?url=https://search-proquest-com.proxyau.wrlc.org/docview/1755486071?accountid=8285.

Somssich, M. (2018) A short history of the CaMV 35S promoter. PeerJ PrePrints. https://doi.org/10.7287/peerj.preprints.27096v1.

Yarris, L. Mystery of Vital Cell Protein Solved After 30 Years. 8 January, 1998. Berkeley Lab. (16 Oct. 2019) < https://www2.lbl.gov/Science-Articles/Archive/3D-tubulin.html>.

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