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Detecting Genetically Modified
Organisms in Every Day Food
2013-2014
GMO Lab Report
By: Zach Obrecht
Abstract
Even with GMOs being one of the most talked about and controversial topics in the food and
agricultural world tod...
Without the labeling, it is extremely difficult to know whether or not a product contains GMOs.
While logical guesses are ...
to the mixture ground it up for a couple more minutes until their sample was mixed with the
DNase water. 50ul of the mixtu...
Results
In figure one, as seen below, two separate gels were made each containing two groups each. For
Preston and Alica’s...
lane is supposed to be negative for GMO DNA which would be found at 200 base pairs, but
positive for plant DNA due to it b...
1 20ul Non-GMO food control 20ul plant master mix
2 20 ul Non-GMO food control 20ul GMO master mix
3 20ul test food 20ul p...
References
Antoniou, M., Robinson, C., & Fagan, J. (2012). GMO Myths and Truths. London: Earth Open Source.
Bryne, P. (201...
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GMO Lab report

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GMO Lab report

  1. 1. Detecting Genetically Modified Organisms in Every Day Food 2013-2014 GMO Lab Report By: Zach Obrecht
  2. 2. Abstract Even with GMOs being one of the most talked about and controversial topics in the food and agricultural world today, it is impossible for the average consumer to see whether or not their food contains GMOs or not. Even in laboratories discovering such information used to be a long and extensive process. But with the help of some modern laboratory technology like gel electrophoresis and thermocyclers, the ability to test a food for GMOs has become possible for common laboratories to test. Our class decided to take advantage of such technology and used it along with restriction enzymes to determine whether or not four common snack foods contained plant DNA and/or GMO DNA. It was found that all four foods tested contained plant DNA. While only two of the four foods contained GMO DNA. Introduction Arguably, one of the most controversial topics in modern agriculture is the use of genetic engineering. Yet while genetically modified (GM) foods are one of the hottest topics today, it is also one of the unknown factors in many foods. It is estimated that 60-70 percent of every food at the grocery store contains at least one GM ingredient (Bryne, 2010). None of these foods, though, are labeled for GM foods by their manufacturers due to the FDA not mandating it. But what exactly are GM foods, why are they made, how are they made, and how they can be detected? In short, GM foods are any foods which have had foreign genes inserted into them. These foreign genes, which come from other plants or animals, give the organisms an enhanced genetic code which modifies the proteins made by the plant. Some foods, for example have foreign genes which allow them to grow in colder temperatures where before they were strictly warm weather plants (Center for Food Safety, 2013). This is just one of the many reasons GM is used with foods today. Historically, the first genetically modified food (or GM food) marketed in the US was the Flavr Savr tomato in 1994- which allowed the tomato to delay ripening after picking (James & Krattinger, 1996). Since then, the industry for GM foods has exploded with the company Monsanto leading the industry. As of 2013, roughly 85% of corn, 91% of soybeans, and 88% of cotton produced in the United States are genetically modified (Center for Food Safety, 2013). According to the FDA and other government organizations which approve these foods, however, there is a reason behind such high percentages. GM foods are said to give a multitude of benefits to the general public and the environment. Some of these benefits include more nutritious food, higher crop yield, a decreased use of pesticides, and disease resistant plants (National Institutes of Health, 2012). But there is another side to the GM food controversy. Aside from potential benefits of GM foods, there are also potential risks to consider. Such risks could include GM foods having potential genetic changes making them unsafe for human consumption and the cause of so called super weeds which have become resistant to many pesticides (National Institutes of Health, 2012).
  3. 3. Without the labeling, it is extremely difficult to know whether or not a product contains GMOs. While logical guesses are possible by comparing the main ingredients of a food with common GMOs crops such as corn and soybeans, they are only guesses nonetheless. However, there is actually a way to detect for the presence of GMOs using modern laboratory techniques. PCR is a molecular technique used to amplify a specific sequence of DNA defined by a set of primers producing millions of copies of that sequence or gene. It does this by rapidly increasing the temperature denaturing the DNA, then lowering it again to allow the primers to anneal to the denatured DNA. The PCR machine then raises the temperature one last time to the optimum temperature for replication forcing the DNA to replicate. Usually, there is around 35-40 cycles per experiment. Gel electrophoresis usually follows PCR amplifications. It is used to separate the amplification product in each sample based on size and charge revealing bands of DNA and their molecular weight. Using these techniques, we conducted an experiment to discover whether or not four common foods contained either GMO DNA or came from a GM plant. Due to the four foods tested--Cheddar Sun Chips, Kettle Corn Chips, Nilla Wafers, and Frito Corn Chips—I expect all four to contain both GMOs. Materials and Methods Two small electronic scales were taken from the back room along with eight weighing trays. The scales were then balanced to ensure the most accurate results and a weighing tray was set on the scale and zeroed out to exclude the weight of the tray from the sample. Our negative control (Bio-Rad Oatmeal) was then weighed out for each of the four groups at the following masses: 1.65 g, 1.5 g, 1.56 g, and 1.52 g. Each group then placed their negative control in a pestle and mortar (1 for each group). The negative controls were done first in order to prevent cross contamination of possible GMO DNA in our test samples. The mass of the negative control was then multiplied by five to determine the amount of DNase water to add. Using this equation groups 1-4, respectfully, added, 8.25 ml, 7.5 ml, 6.45 ml, and 7.6 ml of DNase water to pestle and mortar. The mixture was then ground up for three minutes or until the oatmeal was broken apart. Each group then added the same amount of DNase water as they did the first time once again. The mixture was subsequently ground up for another 3-5 minutes, or until the oatmeal was completely mixed with the DNase water. Following the negative control being mixed in, 50ul of the mixture was pipetted to a microfuge tube filled with 500ul of Instagene. Each group performed this step but with their own sample. The pestle and mortars were then washed out with water and a detergent to attempt to get rid of any trace of the negative control. The scales were zeroed out with a weighing dish again. In chronological order, each group then weighed out their sample to be, 1.5 g, 1.5 g, 1.56 g, and 1.84 grams. Using the same equation mentioned above each group then added, 7.5 ml, 7.5 ml, 7.8 ml, and 9.2ml of DNase water to the pestle and mortar along with their sample food. The order of the previous amounts, from left to right, goes group 1 to group 4. The mixtures were then ground up for a few minutes. The four groups then added the same amount of DNase water
  4. 4. to the mixture ground it up for a couple more minutes until their sample was mixed with the DNase water. 50ul of the mixture was then pipetted into a microfuge tube with 500ul of Instagene. All eight tubes (two from each group) were then placed on a 95 degree Celsius water bath for five minutes before placed in a centrifuge for five minutes at max speed. After making the samples, two PCR master mixes were made using the following ingredients: Mg2+ , dNTPs, PCR water, primers, buffer, and a taq enzyme. The difference between the two types is that the primers for the plant master mix are made to target specific plant DNA; while the GMO master mix primers are made to target GMO genes. Six PCR tubes were then gathered and numbered 1-6. They were then filled according to table 1. The samples were then amplified using polymerase chain reaction inside a thermo cycler set to the following settings: 94o C for 30 seconds 59o C for 30 seconds 72o C for 1 minute Repeat 40 times Following the samples being run through PCR, they were removed from the machine and set into tube holders. Two, 3% agarose gels were then made with 13 cells each. To make each gel, we combined 1.5 grams of gel agarose power with 50ml of 1x TAE buffer in a 200ml beaker. The 1x TAE buffer was made by adding 20ml of 50x TAE buffer with 980ml of dH2O. The mixtures were then stirred until the agarose was completely dissolved and the mixtures were both cloudy and opaque. With the microwave at low power, the two beakers were placed in it and then cooked for 2 minutes. When the gels were removed the mixture had turned translucent. At this stage, ethidium bromide was added to each gel. The gels were then placed back in the microwave for 1 minute. Following the second microwave cycle, the gels were poured into two casting trays. The casting trays themselves had to be taped due to the sides being open which would allow the agarose to pour out the sides. The two gels were then placed in the fume hood and a 13 cell comb was placed into each one. The gels were then given time to polymerize in the fume hood. Following the polymerization, two groups were assigned to each gel. The tape was then taken off the casting trays and the gels were placed in the gel electrophoresis machine. 1x TAE buffer was then added to each machine until the buffer level was higher than the cells. Before the samples were loaded, however, 10ul of Orange G loading dye was added to each of the combined 24 samples. Each of the 13 cells in each gel was then filled 10ul of sample using a micropipette. For both gels, lanes 1-6 and 8-13 contained the samples in numerical order. Lane 7 contained the PCR molecular weight ruler. Following loading, each gel was then analyzed through gel electrophoresis for 30 minutes at 100 volts. The gels were then removed at the end of the 30 minutes and examined under a UV light.
  5. 5. Results In figure one, as seen below, two separate gels were made each containing two groups each. For Preston and Alica’s group (lanes 1-6) their test sample, Frito Corn Chips, tested positive for both plant and GMO DNA. Lanes 8-13 contained Megan and Echoe’s groups’ experiment. Their test food was Nilla Wafers and it tested negative for GMO DNA but positive for plant DNA. In the second gel from lanes 14-19 Casy and Derek’s food sample, Kettle Corn Chips, it too was negative for GMO DNA but positive for plant DNA. Tarence and I’s sample, Cheddar Sun Chips, was analyzed in lanes 21-26 then. The Sun Chips were positive for both GMO DNA and plant DNA. For each sample which tested positive for GMOs as well as the positive control, the bands which appeared were 200 base pairs in length. If a sample was positive for plant DNA or was the non- GMO negative control, bands appeared at 455 base pairs. Primer dimers also occurred in lanes 4, 6, 11, 13, 17, 19, and 26. Discussion Out of the four foods tested, only two of them contained GMO DNA, Sun Chips and Frito Corn Chips. As stated in my hypothesis I expected the Sun Chips and Frito Corn chips to contain GMO DNA, which they did. However, the other two samples, Nilla Wafers and Kettle Corn Chips, were negative for GMO DNA. To me, this came back as surprising due to those two items being derived from big time companies which I assumed would implant GMOs in their products. Due to this I hypothesized that all four food samples would contain GMO DNA. All four samples did contain plant DNA, though. This means that the plant(s) the food item(s) came from were made using genetic engineering and therefore were GM crops. In order to verify the data found for each sample was accurate, we used the DNA ladder found in lanes 7 and 14 along with the positive control(s). For both groups one and four where the food samples tested were positive for GMOs, the band found in those lanes (4&24) aligns with both the GMO positive control (GMO master mix) and the DNA molecular weight ruler at 200 base pairs. The same pattern occurs for the plant DNA which tested positive for all four groups (lanes 3, 10, 16, and 23). Each of them aligns with the GMO positive control (plant master mix) and the molecular weight ruler at 455 base pairs. As stated in the results, primer dimers were seen in lanes 4, 6, 11, 13, 17, 19, and 26. These faint bands, known as primer dimers, occur when two primers anneal with each other during PCR and then are elongated by the taq enzyme. When separated by gel electrophoresis, these two primers are pulled by the positive electrode beyond the DNA ladder and appear as a very faint band. One of the few problems we encountered with this experiment was the non-GMO negative control band not showing up in lanes 1 and 14. Due to it being a non-GMO negative control mixed with the plant master mix, the primers in the master mix were targeting plant DNA. So the
  6. 6. lane is supposed to be negative for GMO DNA which would be found at 200 base pairs, but positive for plant DNA due to it being the target of the primers. However, the plant DNA failed to show up in either of the two gels meaning experimental error must have happened. There are multiple possible answers to this is, one of them being the negative control oatmeal was no ground up enough. Not grinding the oatmeal up enough would not release the DNA. Therefore there was little to no DNA pipetted into the Instagene tubes. There could have also been too much of the sample pipetted into the Instagene tubes (supposed to be 50 ul). Having too much sample would require the Instagene to theoretically ‘work harder’ which would cause the DNases to not be destroyed in the water bath. Failure to destroy the DNases will result in our samples’ DNA being destroyed. Leaving nothing for PCR to amplify and gel electrophoresis to separate.
  7. 7. 1 20ul Non-GMO food control 20ul plant master mix 2 20 ul Non-GMO food control 20ul GMO master mix 3 20ul test food 20ul plant master mix 4 20ul test food 20ul GMO master mix 5 20ul GMO positive control 20ul plant master mix 6 20ul GMO positive control 20ul GMO master mix Table 1. The amount of liquid, in microliters, which went into each of the six PCR tubes. Figure 1. PCR amplification of test samples for the presence of plant- or GMO-DNA sequences. Negative control (lanes 1&2, 8&9, 14&15, 21&22), a DNA ladder (lanes 7&20), and positive control (5&6, 12&13, 18&19, 25&26) samples were processed and amplified in tandem with each test sample. The DNA ladder was composed of five bands measuring 100, 200, 500, 700, and 1000 base pairs in length. The food products tested in the experiment included Frito Corn Chips (lanes 3&4, group 1), Nilla Wafers (lanes 10&11, group 2), Kettle Corn Chips by PopCorners (lanes 16&17, group 3), and Cheddar SunChips (23&24, group 4). For each pair of samples (e.g., 1&2, 8&9 etc.) the first lane was amplified using plant DNA-specific primers while the second lane was amplified using GMO DNA-specific primers. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
  8. 8. References Antoniou, M., Robinson, C., & Fagan, J. (2012). GMO Myths and Truths. London: Earth Open Source. Bryne, P. (2010, September). Labeling of Genetically engineered Foods. Retrieved from Colorado State University: http://www.ext.colostate.edu/pubs/foodnut/09371.html Center for Food Safety. (2013). About Genetically engineered Foods. Retrieved from Center for Food Safety: http://www.centerforfoodsafety.org/issues/311/ge-foods/about-ge-foods Diehl, P. (n.d.). What Are GMOs and How Are They Made? Retrieved from About.com: http://biotech.about.com/od/introtobiotechnology/a/What-Are-Gmos-And-How-Are-They- Made.htm Endelman, R. (2012, March 23). The Difference Between the Terms GM and GMO. Retrieved from The Delicious Truth: http://thedelicioustruth.blogspot.com/2012/03/difference-between-terms-ge- and-gmo.html James, C., & Krattinger, A. F. (1996). Global Review of the Field Testing an Commercialization of Transgenic Plants. ISAAA. Kalinquist. (2012, November 25). What do you know about GMOs? Retrieved from Visualism.org: http://visualism.org/2012/11/25/what-do-you-know-about-gmos-infographic/ National Institutes of Health. (2012, July 5). Genetically engineered foods. Retrieved from MedlinePlus: http://www.nlm.nih.gov/medlineplus/ency/article/002432.htm Various. (2013, November 22). Genetically Modified Food. Retrieved from Wikipedia: http://en.wikipedia.org/w/index.php?title=Genetically_modified_food&action=history

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