Introduction
The purpose of this lab was to test a plant based food sample for the presence of genetic modification. Genetic modification (GM) is the process of altering the DNA in an organism’s genome by “inserting a gene or genes from a donor organism carrying a desired trait into an organism that does not have the trait” (Bawa, A. S. & Anilakumar K. R.), resulting in a genetically modified organism (GMO). Scientists are continuing to find new ways to insert genes for specific traits into plant DNA. A field of massive potential but also “a subject of debate, genetic modification is changing the food we eat and the world we live in” (Ackerman).
GMOs are one answer to the concerning problems of how to produce enough food to feed the growing population and the long term effects of overusing chemicals like pesticides and herbicides, as a main selling point for GM crops is that they reduce the use of pesticides. The primary genetically modified crops grown commercially in fields are “herbicide and insecticide resistant soybeans, corn, cotton and canola” (Bawa, A. S. & Anilakumar K. R.). Other traits of GM crops include increased nutritional value, ability to withstand weather extremes, more fruitful harvests, and even vaccines against infectious diseases (Bawa, A. S. & Anilakumar K. R.). This proposed solution to many current problems has been met with controversy and concern, mostly surrounding human and environmental safety as well as the ethical ramifications. However most people in the United States aren’t even aware of the fact that “more than 60% of all processed foods” (Bawa, A. S. & Anilakumar K. R.) contain genetically engineered ingredients. In this lab, we took a store bought food sample and tested it for the presence of genetically modified DNA.
Hypothesis
Because we wanted the DNA extracted from the food sample to be appropriate, we followed the lab manual’s suggestion of choosing a reliable food with “regards to quality of DNA needed for PCR” (Bio210A Manual, 2017) and decided to test cornmeal. If the cornmeal is a GMO then when analyzing the results of the gel electrophoresis, I would expect to see bands similar in size to those of the positive control with bands amplifying the GM sequence. In order to test our hypothesis, we first extracted DNA from a food sample, then ran PCR reactions to amplify the GMO sequence, and finally used gel electrophoresis to visualize the DNA.
Methods
First we extracted DNA from a certified non-GMO food sample (negative control) and a test food sample that we wanted to analyze for the presence of genetically modified DNA sequences. To prevent any possible contamination while preparing the DNA samples, we prepared the non-GMO sample first. Each sample was weighed and ground with DNase and RNase free water ‘until a soupy mixture was formed’ (Bio210A Manual, 2017). We ground the samples to break apart the cell and release the DNA from the nucleus. Because the DNA was then exposed to DNase enzymes that degrade DNA, we used the InstaGene matrix which “eliminates the cofactors required for DNase function” (Bio210A Manual, 2017). Then they were transferred to tubes containing InstaGene matrix and “placed in a 95℃ water bath for 5 minutes” (Bio210A Manual, 2017) before being spun in the microcentrifuge for another 5 minutes. The microcentrifuge was used to separate the “supernatant (containing the genomic DNA) from the pellet (containing cellular debris and Instagene matrix beads)” (Bio210A Manual, 2017)).
After the DNA had been successfully extracted from the non-GMO food sample and the test food sample, we prepared two PCR reactions for those two samples along with a positive control (certified GMO). After labeling six PCR tubes 1-6, they were kept on ice for the remaining steps. In the first PCR reaction (tubes 1, 3 and 5), DNA samples were added to the plant master mix. The second PCR reaction (tubes 2, 4 and 6) was run with DNA samples added to a GMO master mix. To actually perform the PCR, we placed the tubes in the thermal cycler and used the “GMO-BioRad program (Bio210A Manual, 2017)” .
After completing the PCR reaction we used gel electrophoresis to visualize the products. To cast the gel we combined and heated powdered agarose and TAE buffer. After pouring it into the gel cast tray we placed the comb towards the negative electrode and let the gel solidify. After it set we removed the comb and filled the electrophoresis chamber with more TAE buffer. To prepare the samples we added UView loading dye to each tube and to the molecular weight ruler. Then we loaded samples and the ruler into the gel and started the gel electrophoresis.
Results
PCR reactions identify a specific sequence of DNA, in our case the GM sequence, and makes millions of copies of that sequence, basically “DNA replication in a test tube” (Bio210A Manual, 2017). PCR has three steps: denaturing, annealing, and elongation. To find the specific DNA sequence, the PCR uses primers complementary to the targeted sequence. That sequence is then copied by DNA polymerase. Two PCR reactions were performed on each test food DNA sample. One PCR reaction used primers specific to a common plant gene (plant primers) “to confirm that each sample was derived from plants” (Bio210A Manual, 2017). The other PCR reaction used primers specific to sequences commonly found in GM crops (GMO primers). This PCR reaction only amplified DNA if the test food was GM.
As shown in fig. 1, the test food gave a positive result with both the plant and GMO primers (lanes 3 and 4). A non-GMO sample would’ve only given a positive result with the plant primers. If the test food was non-GM, then the “GMO primers would not be complementary to any sequence within the test food genomic DNA and would not have annealed so no DNA would be amplified” (Bio210A Manual, 2017).
To find out whether DNA had been amplified or not, the PCR products were electrophoresed on a gel and stained to visualize DNA as bands. Gel electrophoresis separates DNA fragments according to size and charge. When an electrical current is applied to the gel, the DNA fragments are transported through the gel with the smaller fragments traveling faster and the larger fragments taking much longer. A molecular weight ruler was electrophoresed with the samples to allow the sizes of the DNA bands to be determined. Because DNA is a negatively charged molecule, the comb was placed at the negative end so that the molecules will migrate to the positive electrode.
In the results of the gel electrophoresis shown in fig. 1, lanes 1, 3 and 5 all have bands of approximately 500 bp. Lanes 4 and 6 have bands that are approximately 200 bp. Lanes 1 and 2 show what the test food bands (lanes 3 and 4) would look like if they were non-GMO while lanes 5 and 6 show what the bands in lanes 3 and 4 would look like if they were GM.
Conclusion
The purpose of this lab was to test cornmeal for the presence of genetic modification by isolating the genomic DNA from the food and then conducting a PCR reaction to amplify the genetically modified DNA as well as control DNA. Then by running the results of the PCR through a gel electrophoresis we were able to determine that the data does support our hypothesis and the test food does contain genetic modifications.
In this lab we had positive and negative controls. We were able to determine that the cornmeal is a GMO by looking at the bands in the gel electrophoresis. Lanes 1 and 2 were the negative control (non-GMO), lanes 3 and 4 were the test food (cornmeal), and lanes 5 and 6 were the positive control (GMO positive DNA). If our test food was not GM then we would’ve seen bands in lanes 3 and 4 similar to those in lanes 1 and 2. However, because our test food is GM, we see bands in lanes 3 and 4 similar to those in lanes 5 and 6 (the GMO positive DNA). We used a negative control to ensure that no contamination from the GMO positive DNA got into the test food as that would’ve led to false results. The positive control was used to show what we should expect to see from our test food if it was GM.
The other bands represented in Fig. 1 that are approximately 100 bp are primer dimers. Primer dimers (PDs) are common byproducts of PCR and are usually visible after gel electrophoresis. Primer dimers occur when primers used in PCR “bind to themselves or to each other instead the template DNA because of complementary bases in the primers (LB-145 Laboratory Manual, 2011). This results in primers acting as “their own templates to make a small PCR product” (LB-145 Laboratory Manual, 2011) and is why they do appear on gel electrophoresis. Because primer dimers result in the complementary bases in the sequences, the best way to avoid PDs is to “make sure that there are few complementary areas in the base sequence of the primers” (LB-145 Laboratory Manual, 2011) where they would be able to bind to each other instead of the template DNA.
Because every group tested a different food sample, results will vary in lanes 3 and 4. However, the bands in lanes 1, 2, 5, and 6 should be similar as those were the control lanes. Variations in those bands would be due to contamination of the DNA samples. While performing the lab we were cautious to avoid any cross contamination by preparing the non-GMO samples first and keeping a clean environment.
Other background research has shown that GMOs are “more highly regulated than other foods that we eat” (Bawa, A. S. & Anilakumar K. R.). They are thoroughly screened and tested as well as monitored to ensure that they are “substantially equivalent to conventional foods” (Ackerman). GMOs have potential to “eliminate world hunger and better the lives of all” (Ackerman) but they still carry risks known and unknown. However, if we remain cautious about how and why we implement these genetically modified foods, and if we continue to be vigilant about testing them thoroughly, GMOs have the potential to be one tool in the toolbox that can help solve these problems.
References
- San Diego Mesa College. (2017). Bio210A Laboratory Manual 8th Edition. San Diego, CA
- Michigan State University. (2011). LB-145 Laboratory Guide 8th Edition. East Lansing, MI
- Bawa, A. S. & Anilakumar K. R. (2013). Genetically Modified Foods: Safety, Risks, and Public Concerns. Journal of Food Science and Technology. 50(6), 1035-1046.
- Ackerman, Jennifer. Food: How Altered? National Geographic.