Genetically Modified Organisms INTRODUCTION: The purpose of this lab was to identify if non-labeled food products are actually genetically modified foods. Before we could begin testing this theory we first had to gain an understanding about genetically modified organisms in general. This was rather easy because if you have been to any grocery store lately you have without a doubt seen products with labels saying “GMO-free” or even “contains only non-GMO ingredients. ” GMO actually stands for Genetically Modified Organisms, and this refers to any products that have been manipulated or altered at the gene level.
Modifying plants is not a new concept. “For centuries, gardeners and farmers have been crossbreeding different species of plants to create plants that produce heartier, better tasting, or more beautiful crops. ” (Mateljan) However, crossbreeding of plants is acceptable to most individuals where as genetically modifying foods is not. According to the paper Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize ” Currently, no regulatory authority requests mandatory chronic animal feeding studies to be performed for edible GMOs and formulated pesticides. (Seralini)This is a major concern to many individuals who have been unaware that they were consuming GMO’s in the first place. The genetic engineering of foods that is called GMO’s causes many concerns around the world. To genetically engineer a plant one must first obtain genes from an animal, plant, bacterium, or virus. These genes are then inserted into a different organism, usually a plant, which changes the genetic code of the plant forever. The genetic code, or the blueprint that is used to determine all of the organisms physical characteristics are changed within the organisms, this is where people tend to have a problem with GMO’s.
According to the paper How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed ” In many countries legislations concerning GMO commercialisation have been adopted and although they differ from country to country, some issues are common. ” (Sylvia R. M. Broeders) But, does it make the process by which these new foods are made? With this biotechnology many scientist have been able to create tomatoes that last longer on store shelves, soybeans that are resistant to weed killers, potatoes that can produce pesticides, even make glow in the dark fish and puppies. Not all things that happen with GMO’s are bad.
Scientist are working to make fruits, vegetables, and even grains that are higher in levels of vitamins and nutritional value. There is even talk of vaccines that can fight diseases being produced in food as well. Many people have voiced concerns over food allergies and toxins that may be present in GMO’s. At this point in time it is unknown what the health risk are when it comes to consuming genetically altered foods. And, with very few studies that have been performed to find out how these GMO foods effect human health we may never fully know if there is a health risk involved.
That is why this lab is important. No matter what position, for or against, a person is for the consumption of GMO foods it is beneficial to all of us to be able to test to see if GMO’s are present in the food we eat. To do so we learned about two methods, enzyme-linked immunosorbent assay, or ELISA, which was not useful in our lab experiment because the foods we were testing were very highly processed and the proteins were most likely destroyed. Instead we used polymerase chain reaction, or PCR to look for DNA sequences that are common in GMO foods.
My hypothesis for this experiment was that we would be able to detect GMO’s in most if not all of the processed foods that we tested. MATERIALS AND METHODS: In the first part of the experiment we needed to extract DNA from different food samples. In the second part of the lab we ran PCR reactions to amplify GMO and natural plant sequences from the DNA, in the third part of the experiment we electrophoresed the amplified samples to visualize the DNA. To perform each step listed above we needed certain reagents for each process for DNA Extraction we needed a control non-GMO food sample and a testable food sample.
Water and a mortar and pestle to grind the sample. Insta gene Matrix was added to two tubes and a water bath. To perform PCR Reactions we needed DNA template that were going to amplify, DNA polymerase, two DNA primers, four DNA base pair subunits and buffers. For electrophoresis we needed PCR product that we collected, molecular weight ruler, and fast blast for staining. DNA Extraction- For this experiment we used blueberries as our possible GMO food and were given a non-GMO of certified grain as our Non-GMO test sample. Before handling any samples we first needed to label two screw cap tubes with our names, date, and non-GMO and test.
After labeling 500? l of InstaGene matrix was added to each tube. A mortar and pestle, that was cleaned with a mixture of bleach and water was used to maintain a sterile environment for the non-GMO food. Weighed out 1. 95 g of non-GMO certified grain and placed them in the mortar. Using a transfer pipet added 5ml of distilled water for every gram of food. (See calculations part 1) Grinded the non-GMO certified grain with a pestle for 2 minutes until a slurry formed in the mortar. Added another 5ml of distilled water for every gram of food, mixed until it was smooth enough to pipet.
Removed 50? l of the slurry and placed it into the screw cap tube that already contained the 500? l of InstaGene Matrix labeled non-GMO. Recapped the tube, shook well. Washed the mortar and pestle with a mix of bleach and water to make sure it was clean for the test food. Weighed out 1. 90g of blueberries(test food 2) and placed into the mortar. Used a transfer pipet and added 5ml of distilled water for every gram of food. (See calculations part 2) Grinded the blueberries with a pestle for 2 minutes until a slurry formed in the mortar.
Added another 5ml of distilled water for every gram of food, mixed until it was smooth enough to pipet. Removed 50? l of the slurry and placed it into the screw cap tube that already contained the 500? l of InstaGene Matrix labeled test. Recapped the tube, shook well. Washed the mortar and pestle with a mix of bleach and water to make sure it was clean for the test food. Weighed out 1. 95g of peanuts(test food 1) and placed it into the mortar. Used a transfer pipet added 5ml of distilled water for every gram of food. (See calculations part 3) Grinded the peanuts with a pestle for 2 minutes until a slurry formed in the mortar.
Added another 5ml of distilled water for every gram of food, mixed until it was smooth enough to pipet. Removed 50? l of the slurry and placed it into the screw cap tube that already contained the 500? l of InstaGene Matrix labeled test. Recapped the tube, and shook well. Washed the mortar and pestle with a mix of bleach and water and put it away for later use if necessary. Turned on the heat block and set the temperature to 95 °C. Once the heat block reached the appropriate temperature we placed both samples labeled non-GMO and test onto the heat block for 5 minutes starting at 10:05am and ending at 10:10am.
Placed both tubes into a centrifuge machine and spun for another 5 minutes at max speed. Stored the tubes in the refrigerator till the next lab. PCR Reactions- To begin we needed to label eight PCR tubes with our initials, date, and sample. (See table 1 for labels) Using a fresh pipet for each tube we added 20 ? l of green plant master mix to tubes 1,3,5,and 7. Added 20 ? l of GMO master mix to tubes 2,4,6, and 8. Capped each tube to keep sample uncontaminated. With a new pipet for each tube added 20 ? l of non-gmo sample to tubes 1 and 2. Added 20 ? l of test food 1 to tubes 3 and 4, added 20 ? of test food 2 to tubes 5 and 6, and last but not least added 20 ? l of GMO positive control to tubes 7 and 8. Placed all 8 samples into the thermal cycler for the indicated processing time in table 2. Electrophoresis- We needed and had to setup an electrophoresis chamber as pictured in image 1. And a 3% Agarose gel that we cast inside the electrophoresis chamber. To make the gel we used 20 ml of 10 XTBE and added it to 180ml of water to dilute the 10 XTBE to 1 XTBE. Used 50 ml of 1 XTBE and added 0. 5 grams of Agarose to the 1 XTBE and microwaved the mixture until all sediment was gone.
Cooled the mixture to room temperature and poured it into the designated chamber inside the electrophoresis setup. Waited until it solidified and then removed the comb and poured additional 1 XTBE over the gel and filled the side chambers of the electrophoresis setup. Returned to the 8 samples after setting up the electrophoresis chamber and started adding 10 ? l of Orange G loading dye to each tube and mixed well. Loaded the gel with 20 ? l of ladder and sample in the order listed in table 3 under Results and Data Electrophoresis. Ran the gel at 100 volts for 30 minutes. Stained the gel using fast blast.
To stain the gel we used a quick stain protocol. Removed the gel and placed it into a tray and poured 100x Fast Blast on top of the gel till it covered the gel. Let it sit for 5minutes with slight agitation and then transferred the gel to another tray to wash the gel with warm water for 10 seconds. Emptied the water and refilled the tray with fresh water and let it sit for 5 minutes three additional times. Viewed our gels and read our results. (see figure 2 for gel. ) However, as a class we all used the same procedures as listed above on different food samples, there was data collected and that is shown in figure 3.
Figure 1: Gel Electrophoresis Chamber Setup This is a photo of the setup of the gel electrophoresis chamber and what it should look like if set up properly RESULTS AND DATA: DNA Extraction Calculations Calculations part 1: Mass of food = ____ g x 5 =____ ml Non-GMO certified grains1. 95 g Mass of food = 1. 95 g x 5 = 9. 75 ml of water Calculations Part 2: Mass of food = ____ g x 5 =____ ml Blueberries1. 90 gr Mass of food = 1. 90 g x 5 = 9. 50 ml of water Calculations Part 3: Mass of food = ____ g x 5 =____ ml Peanuts 1. 95 g Mass of food = 1. 95 g x 5 = 9. 75 ml of water PCR Reaction Tables
Table 1: Tube numbers and what mix they contained This table shows the tube numbers and what they contain Tube #| DNA| Master Mix| 1| Non-GMO| Plant Mix| 2| Non-GMO| GMO Mix| 3| Test Food 1| Plant Mix| 4| Test Food 1| GMO Mix| 5| Test Food 2| Plant Mix| 6| Test Food 2| GMO Mix| 7| Positive GMO| Plant Mix| 8| Positive GMO| GMO Mix| Table 2: Thermal Cycler Set Up- This table shows the thermal cycler settings needed for this PCR reaction 94°C| 2 minutes | 94°C59°C 72°C x 40 cycles| 1 minute1 minute2 minutes| 72°C| 10 seconds| 4°C| ? | Table 3: Loading order for gel 1| 2| 3| 4| 5| 6| 7| 8| 9|
Non-GMO(neg)| Non-GMO(neg)| Test Food 1(plant mix)| Test Food 1(GMO mix)| Positive Control Plant| Positive Control GMO| PCR MW Ruler| Test Food 2 (Plant mix)| Test Food 2 (GMO mix)| Figure 2: Completed Gel from our samples No Data collected Figure 3: Class data collected from gels DISCUSSION: The object of this lab was to utilize the method of PCR to detect GMOs in our food samples. The presence of GMOs in a sample was indicated by a band in the agar gel. We expected to see a band around the 1000 base pair ladder to use for size extrapolation. Another band around the 500 base pair area to show it is a plant gene.
And a band around the 200-300 base pair area if the food has been genetically modified. After our PCR was done we obtained the following results for the GMO found in our food samples. On our gel in particular there were no bands found at all. This could be because we had to leave the gel for an extended amount of time in the refrigerator before we were able to actually stain and read the gel. It is believed that the DNA that was present degraded over the week that we had off from school. So when we stained the gel there was nothing left to view. However, as a class we had 17 total food samples.
And, out of the 17 total food samples nine of our samples had a GMO band (figure 3). This would leave us to believe that GMO’s were indeed present in our samples even though we had no bands on our gel. From the chart we can see that corn was determined to be GMO negative, but still showed a band for having plant DNA. We could also see that the corn meal/corn flour has no bands in either column. This shows that it did not contain GMO’s, but it also says that it’s not made of plant material. This could be because the corn meal/corn flour was so over processed that most of the real corn DNA may have been removed.
For the Cheeto’s it is shown that there is a band for plant DNA which we suspected would happen because they are made from corn. However, there is conflicting data that shows a band in the GMO lane. I would have to read this as positive for the presence of GMO’s even though the bands look very faint in two of the images. For the peanuts it shows a band for plant DNA which we expected to see and no band for GMO’s which we also expected to see since the peanuts are not a processed food. Last but not least are the grits, we can see a band for plant DNA as well as a band for GMO DNA.
Again this was an expected result since grits are a highly processed food made from many plant materials. In conclusion it is fair to say that our experiment involving the use of non-GMO samples and what we presumed to be GMO samples was conclusive in that foods that are highly processed tested positive for GMO DNA while foods that were unprocessed showed no signs of GMO DNA but still showed signs of plant dna. This experiment although conclusive could have been performed at a better level if we had only used one organic sample and one non-organic sample.
That way we could determine if organic food really is organic or if GMO’s are still present in what is considered to be organic foods. We also needed to be able to perform the experiments back to back. We had too much lag time in between experiments and lost much of the needed data that we would have been able to collect had we been able to proceed without large daily gaps in the schedule. I think this experiment was successful in showing that GMO DNA is present in processed foods. If I had to improve upon this experiment I would limit the samples that could be tested.
I would also try to make sure that the time in between viewing gels is not as long. We might have been able to see a result if a week had not passed by before we were able to view the gels. Works Cited Mateljan, George. The Worlds Healthiest Foods. Seattle: George Mateljan Foundation, 2006. Seralini, Gilles-Eric. “Long term toxicity of a Roundup herbicide and a Roundup-tolerant. ” Elsevier (2012): 4221-4231. Sylvia R. M. Broeders, Sigrid C. J. De Keersmaecker, and Nancy H. C. Roosen. “How to Deal with the Upcoming Challenges in. ” Journal of Biomedicine and Biotechnolog (2012): 11.