Lab 12 - GMO Food Samples PDF

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas

BIO 101 Lab 12: Investigating GMO Status of Food Samples Notification: If you have a disability that makes it difficult to complete this lab, please contact your instructor. Please provide your instructor a copy of the Memorandum of Accommodation (MOA) from NVCC Disability Support Services. Objectives: x Explain the term GMO x Describe the function and main steps of PCR x Explain the roles of the positive and negative controls of the experiment x Analyze gel electrophoresis data to identify GM positive and GM negative food Background: The United States Department of Agriculture (USDA) defines Genetically Modified Organism (GMO) as “an organism produced through genetic modification”. Genetic Modification is defined as: “the production of heritable improvements in plants or animals for specific uses, via either genetic engineering or other more traditional methods. Some countries other than the United States use this term to refer specifically to genetic engineering.” Finally, Genetic Engineering is defined as: “manipulation of an organism's genes by introducing, eliminating or rearranging specific genes using the methods of modern molecular biology, particularly those techniques referred to as recombinant DNA techniques.” https://www.usda.gov/topics/biotechnology/biotechnology-glossary There are many reasons why organisms have been genetically modified. Goals of such bioengineering include resistance to pathogens, improved nutritional content, resistance to herbicides, increased rate of photosynthesis, and increased stress resistance. In the US, GMO plant species currently in widespread production include corn, soybeans, squash, cotton, canola, and sugar beets. Many of the GMO products are foods that we consume daily. For example, canola oil is widely used in food products, and almost all US grown canola is genetically modified. The same is true for soybeans - over 85% of US soybeans are genetically modified. There is no requirement in the US that GMO foods be labeled as such and no verification that foods that are labeled as “GMO-free” actually have absolutely no genetically modified content. However, it is possible to test foods using a technique called Polymerase Chain Reaction (PCR) to determine if they do or do not contain GMO material. Introduction to PCR The polymerase chain reaction (almost always simply referred to as PCR) was developed by Kary Mullis and colleagues in 1983. Mullis envisioned a method to replicate specific pieces of DNA in such a way that the target DNA was amplified. Then, as now, a cycle of PCR included three stages:

BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas 1. Denaturation. The template DNA is heated WRÛ&WRGHQDWXUHWKHVWUDQG5HPHPEHU that the enzyme DNA polymerase synthesizes a new strand of DNA as it moves along a single stranded template.) 2. Annealing. The reaction temperature is dropped to 50–Û& WR DOORZ WZR SULPHUV WR anneal to the template DNA. Primers are designed to stick to one end or the other of the target DNA sequence. 3. Extension7KHUHDFWLRQWHPSHUDWXUHLVUDLVHGWRÛ&DQG'1$SRO\PHUDVHXVHVIUHH nucleotides to synthesize a new strand complementary to the template DNA. The three stages are repeated for 30–40 cycles. At the end of this process, the number of copies of the target sequence is several orders of magnitude greater than at the beginning of the reaction. One important technological development in the history of the PCR technique was the invention of the thermocycler. Initially, a the temperature of a sample was changed to allow denaturation, annealing, and extension to happen by manually lifting the reaction tubes from one water bath to the next every few minutes for several hours (remember that PCR involves 20–40 cycles). However, in the late 1980s, an instrument (the thermocycler) was developed that automated these temperature changes. Thermocyclers have a metal block that holds sample tubes and the temperature of the block can be raised and lowered very rapidly. The reaction tubes also have very thin walls to facilitate rapid temperature changes occurring in the samples themselves. As a result, PCR can now be done in only a few hours without constant supervision by simply putting reaction tubes in a thermocycler and pressing a few command buttons. Initially, performing PCR also required that more DNA polymerase enzyme be added to the reaction after each denaturation step because heating the reaction to the temperature required to denature DNA also permanently denatured the DNA polymerase. However, DNA polymerases had been discovered in organisms that live in the thermal pools at Yellowstone National Park — VLQFH WKH EDFWHULD OLYHG LQ !Û& water, their enzymes had to function at those temperatures. A DNA polymerase isolated from the bacteria Thermus aquaticus (called Taq polymerase) made PCR the common tool we know today (although other heat stable DNA polymerases have since been discovered).

BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas Using PCR to determine if food samples contain GMO material There are a number of genes or DNA sequences commonly added into organisms to make them into GMOs. Using PCR and primers that can specifically bind to these genes or DNA sequences (if they are present), we can determine whether or not a food sample contains geneticallymodified material due to the primers enabling amplification of DNA from many (though not all) GMO foods. In this activity, we will set up two separate PCRs for each food sample. The first reaction will use genetic modification-detecting primers (present in the GMM reagent) If PCR with these primers results in amplification, that may indicate the sample contains GMO material. If PCR with these primers does not result in amplification, it could be because of several reasons: (1) the food is not genetically modified, (2) the food is genetically modified but does not contain genes recognized by the genetic modificationdetecting primers, or (3) the PCR did not work. We have no way to control for the second possibility, but to control for the third, a second PCR can be run on each sample using general plant-detection primers (present in the PMM reagent). These primers recognize a plant gene (specifically the Photosystem II gene from chloroplasts). If the PCR using the general plant-detection primers produces a product and the PCR using the genetic modification-detecting primers does not, then you know that the PCR procedure worked and that the food sample does not contain GMO material. If the PCR using the general plant-detection primers produces a product and the PCR using the genetic modification-detecting primers also produces a product, then you know that the PCR procedure worked and that the food sample does contain GMO material. If the PCR using the general plant-detection primers does not produce a product, then regardless of the results of the PCR using the genetic modification-detecting primers, you should assume that the PCR procedure did not work as expected. This unfortunately means no conclusion can be drawn regarding the GMO status of that food sample. Reasons why the PCR may not work include the starch present in some food samples (such as fresh corn) inhibiting the activity of Taq DNA polymerase, which would prevent PCR from taking place. Activity Overview The steps in this experiment are choosing your samples, grinding the samples and extracting DNA, running PCR on the samples, and analyzing the results with agarose gel electrophoresis. Choosing your sample. If you want to find foods that contain genetically-modified material, you will be most successful if you choose samples that contain soy or corn. Many corn products, such as corn meal, grits, and corn chips are likely to have genetically-modified content. (Surprisingly enough, processed foods work quite well in this experiment. DNA survives BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas whatever it takes to turn corn into corn chips.) Soy products such as vegetarian “meats” are also likely to have genetically-modified content. Avoid oils (DNA will not be present in oils), and heavily processed foods like cornflakes, potato chips, and fries. Fresh products should work, but the starch in corn and soybeans interferes with PCR. Papaya from Hawaii (if you can find any from Hawaii) is also likely to be genetically-modified. You may also wish to test foods that are labeled as “non-GMO” or “Organic” to see if you can find genetically-modified content in those foods.

Examples of foods available for testing

Positive and negative controls (from corn meal and certified Genetic Modification-free oatmeal, respectively) will be provided to you to compare to your experimental samples. Grinding the samples and extracting DNA. You will grind each sample in water in a mortar and pestle. After the sample is ground well, a small amount will be mixed with InstaGene matrix. InstaGene is a suspension of tiny negatively charged beads that bind cations. Removing cations such as Mg2+ from the DNA will prevent the activity of enzymes that could degrade the DNA. After the InstaGene matrix is pelleted by centrifugation, the supernatant (top layer of liquid) will contain the genomic DNA.

PCR of the samples. The materials required for each reaction are mixed in PCR tubes and placed in a thermocycler to perform PCR. The following components of each reaction are premixed as part of the PMM and GMM reagents: x primers x reaction buffer

x dNTPs (a mixture of dATP, dTTP, dCTP, and dGTP nucleotides) x Taq DNA polymerase

The genomic DNA extracted from the food samples will be added to these pre-mixed components.

BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas The thermocycler will be programmed by the lab staff or lab instructor as follows: Purpose Initial denaturation PCR Final extension Hold

Temperatu re Û& Û& Û& Û& Û& Û&

Time 2 min 1 min 1 min 2 min 10 min 

Number of Cycles 1 40 1

Gel analysis. To visualize the results of the PCR, we will use agarose gel electrophoresis. Premade positive (+P and +G) and negative (–P and –G) control reaction tubes will be provided to you. These, along with your food sample reactions will be loaded onto an agarose gel. The product from the plant primers should have a size of approximately 450 bp, while the product from the GMO primers (if present) should have a size of approximately 200 bp. Using the positive and negative controls, you will analyze your gel and determine whether your test food sample contains genetically-modified content. See the gel image below for an example.

BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas Materials: x

Internet

Safety: Follow all standard laboratory safety procedures. Do not rinse food particles down the drain. Procedure: 1. Read the following National Geographic Encyclopedic entry on Organelles. https://www.nationalgeographic.org/encyclopedia/organelles/ 2. In the laboratory, you would perform the following steps to extract DNA from a food sample: a. Wash a mortar and pestle well with soap and water at the sink and rinse them with distilled water. Spray the mortar and pestle with 10% bleach, let sit for 2 min, and rinse with distilled water. Dry them with a paper towel. b. Obtain 1 tube of InstaGene matrix and label the tube with your group members’ initials. Note: label the tube itself, in case the lid gets separated from the tube. c. Weigh out approximately 1 g of the test food sample using the weigh boat and put the sample in a clean mortar. d. Add 5 ml of distilled water per 1 g of sample to the mortar and grind the sample with the pestle until it forms a slurry. This usually takes at least 2 min. e. Add 25 ml of distilled water to the mortar and continue to grind until the sample is smooth enough to pipette. f. You would then transfer 50 µl (microliters) of the food slurry to the InstaGene tube using a 50 µl micropipette. g. Watch the following video about using a micropipettor from time 0:00 until 4:22. https://www.youtube.com/watch?v=p-OPOYbeZP0&t=2s h. Next, you would wash the mortar and pestle well with soap and water at the sink and rinse them with distilled water. Spray the mortar and pestle with 10% bleach, let sit for 2 min, and rinse with distilled water. Dry them with a paper towel.

BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas i. Then, you would mix the contents of the “Instagene” tube briefly by vortexing, IROORZHGE\LQFXEDWLQJWKHVDPSOHDWÛ&IRUPLQ j. You would then centrifuge the sample in the microcentrifuge for 5 min at max speed, being sure to balance the microcentrifuge. You may need to coordinate with another group to do this. k. After this, you would identify your PCR mastermix tubes containing 5 µl Plant Mastermix (labeled “PMM”, colored green) and 5 µl GM Mastermix (labeled “GMM”, colored red). l. Then, you would label the PCR tubes with your group members’ initials and section number. Note: label PCR tubes on the sides, because the heated lid of the thermal cycler often ruins writing on the tops of the tubes. m. Next, you would transfer 10 µl of your food DNA test sample to each PCR tube. Be very careful not to disturb the InstaGene pellet — if you do, pellet the InstaGene again before continuing. n. Mix the PCR tube contents by vortexing. o. Finally, you would centrifuge the samples using the microcentrifuge and wait for your instructor to collect them and place them in the thermocycler to perform PCR. 3. To see all of the steps described above, watch the following video showing how to obtain DNA from foods for the purpose of testing whether or not the foods are Genetically Modified Organisms (GMOs) from time 0:00 until 6:37. https://www.youtube.com/watch?v=RIPsjrXMMmw Note that in the video there are two reactions being set up for each DNA sample. The explanation for why those two reactions are being done is below. The reaction containing the PMM (green color) mixture is being performed to detect the presence of Plant DNA (specifically chloroplast DNA) in the sample. If the results for the PCR using the PMM mixture indicate that there is not any Plant DNA in the sample, then we would know something went wrong with the process of either obtaining the DNA or the process of detecting it using PCR. The reaction containing the GMM (red color) is being performed to detect the presence of foreign DNA (inserted due to genetic modification). If the results for the PCR using the GMM mixture indicate that there is foreign DNA in the sample, then we would know that the food contained cells from Genetically Modified Organisms. If the results for the PCR using the GMM mixture indicate that there is not any foreign DNA in the sample, BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas then we would be fairly confident that the food did not contain cells from Genetically Modified Organisms. 4. Watch the following video that describes the process of Polymerase Chain Reaction (PCR) ands its uses from time 0:00 to time 1:09 and from time 1:38 to time 10:12. https://www.youtube.com/watch?v=uKeMiAZ8Zu4 5. Watch the PCR Animation from time 0:00 to time 3:31 for an additional depiction of the process of PCR. https://www.youtube.com/watch?v=DkT6XHWne6E 6. Based on the Background information, the National Geographic Encyclopedic entry, and the videos you watched, answer Questions 1-2 on the Lab Worksheet 7. For the lab activity, two types of corn chips were analyzed to determine if they contain GMO corn or not. The image below shows the corn chips that were tested. These were Harris Teeter Organics Corn Chips (USDA Organic Labelled) and Tostitos Corn Chips (not labelled as organic). Answer Question 3 on the Lab Worksheet, by writing a hypothesis for each of these two types of corn chips about whether or not you think those chips contain GMO corn.

8. After the PCR has been completed, you will analyze the results using agarose gel electrophoresis by performing the following steps: Note that in a typical laboratory activity, the PCR would continue for several hours and the samples will be removed from the thermal cycler by lab staff and stored at –Û& until the following week, when you would continue the lab activity by analyzing the results using agarose gel electrophoresis.

BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas a. You would position the gel box so that the electrodes are on the side away from you, and place the 2% agarose gel into the gel box so that the wells are to the left. b. Then, you would fill the gel box with SB running buffer to the fill line or until the

gel is covered. c. Next, to prepare your PCR samples for electrophoresis, you would add 5 µl of loading dye to each sample and mix by pipetting gently up and down. d. You would also obtain positive and negative control reaction tubes and add 5 µl of loading dye to each of them. e. Then, you would briefly centrifuge your tubes so that the contents are pulled to the bottom. f. Next you would load 10 µl of each sample in the following order: Lane

Sample

1

Negative control (non-GM) with plant primers (-P)

2

Negative control (non-GM) with GMO primers (-G)

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Unknown test sample with general plant-detection primers (PMM-T)

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Unknown test sample with genetic modification-detection primers (GMM-T)

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Positive control (GM content) with plant primers (+P)

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Positive control (GM content) with GMO primers (+G) g. Finally, you would connect the electrodes to the power supply according to color and run the gel at 150 volts until the yellow marker is between 5 and 6 cm on the tray. This may take about 45 min.

9. To see the process of agarose gel electrophoresis, watch the following video showing how to separate DNA fragments from each other using agarose gel electrophoresis from time 0:00 to time 4:06 https://www.youtube.com/watch?v=vq759wKCCUQ&t=135s 10. To complete the gel electrophoresis and obtain an image of the DNA fragments in the agarose gel, you would perform the following steps:

BIO 101 Lab 12: Investigating GMO Status of Food Samples

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To submit this assignment, students will complete the Lab Worksheet on pages 11-13, then upload their completed document as a DOC or PDF file in Canvas a. First, turn off the power supply and unplug it. b. Next, carefully remove the casting tray from the electrophoresis chamber. Be careful not to let the gel ...