Detecting Genetically Modified Organisms Lab Report

Title: Detecting Genetically Modified Organisms Introduction: The debate between the pros and cons of using genetically modified organisms (GMO) have greatly been discussed for many years. A GMO is a living organism whose genetic material is manipulated by inserting another organism’s DNA into their genome. Not only can a genetically modified crop grow faster, but it can even be more nutritious. People around the world think that the answer for curing world hunger is GMO crops that can be produced in larger quantities. Some other pros for GMO’s in agriculture are crops bring insect and pesticide resistant. Although there are many benefits that GMO’s bring to the table, there are also some troubling cons that have been brought up as well. A common GMO misconception is that they can provoke an allergic reaction, but that is not the case because one is born with an allergy. Another misconception is that GMO’s can transfer genes to the consumer, but again, this idea is not supported by any facts. A more possible con of GMO’s is outcrossing. Outcrossing is the movement of genes from a genetically modified plant into a conventional crop or surrounding wild species. A strategy that is being used to keep this from happening is clearing separating the fields of GMO and conventional crops to prevent the movement of genes. A major concern that people having around the possibility of GMO’s outcrossing is the ability of pest or herbicide resistance spreading to weeds. In this experiment the GMO status of 3 common foods (pine nut, corn, and soy) were determined by first determining the genotype of each food. The are 2 base pairs that are being focused on. The base pair 162 product (35S/NOSt) is a bacterial gene which will determine if a sample is a GMO. The base pair 187 product is tubulin which is found in all plants and should be seen in all samples in the experiment. These base pairs will be isolated and amplified by a polymerase chain reaction (PCR). PCR occurs when a DNA sample that includes a primer, DNA polymerase, and other necessary components are heated up at specific temperatures. The sample are at these temperatures for a set amount of time where it undergoes denaturing and annealing processes until there is an abundance of copies of the DNA sample. If the corn mufÏn mix shows bands on bp 162 and 187, then that food is a GMO. Methods and Materials: Step 1: Prepare sample food test tubes The 3 foods used in the lab were pine nut, corn mufÏn mix, and soy chips. All of 3 foods started off in their normal solid form, so the first step is to grind them down into a powder. A different pestle and paper cup were used to ground down each of the foods to ensure that the samples would not be cross contaminated. After a cohesive fine grain powder was obtained, .1 ml of each powder was added to 3 separate labeled test tubes. This was done by scraping the powder in the tube using different micro pestles and filling. The line towards the base of the tube was used to judge how much powder to fill. After pressing down the powder in each of the tubes, 400 microliters of DNA reaction buffer were added into all 3 test tubes. When dispersing the buffer in each of the tubes, the tip of the pipet hovered above the test tube opening to prevent cross contamination. Using the same corresponding micro pestle from before, each of the mixtures in the tubes were mixed until no solid debris could be seen. To further mix the samples each tube was vortexed for 30 seconds. Following the vortex the tubes were incubated for 15 minutes for 56°C. After the incubation 300 microliters of sodium chloride was added to each tube to neutralize and promote separation. Like before, to mix the tubes they were vortexed for 30 seconds. Next, the tubes were placed in the centrifuge at full speed (13.4 rpm) for 5 mins. When placing the tubes in the centrifuge the tubes were opposite of one another with hinges facing outward. After the centrifuge, the solid debris fell to the bottom and the supernatant is what is needed in the experiment. 500 microliters of each of the supernatants were dispersed in new labeled tubes. When drawing in the supernatant into the pipet, the plunger was pressed down before being inserted into the liquid making sure now to draw in any solid pieces. 500 microliters of isopropanol was added to each of the supernatants, vortexed and then let sit for 5 minutes at room temperature. After being let sit, the tubes were centrifuged for 5 minutes which formed a small pellet at the bottom of the tube. To prepare the DNA of each of the foods is to remove the supernatant in the tube leaving the pellet that contains DNA in the tube. To remove the supernatant the pipet was set at 500 microliters and after drawing in the liquid, it was disposed of into a waste beaker. To make sure the pellet containing the DNA is the only substance left in the tubes, 500 microliters was added down the sides of each of the tubes and then run at full speed in the centrifuge for 2 minutes. The liquid in the tubes were decanted and the tubes were left open to dry. Finally, 50 microliters of DNA buffer were added to preserve the DNA and the tubes were frozen. Step 3: Performing PCR To begin the process of PCR the first step was to resuspend the primer mix with 1000 microliters of Tris EDTA (TE buffer). To mix thoroughly the tube was flicked and then placed in the bench top cooler. Next, the PCR positive control needed to be resuspended using 190 microliters of the TE buffer. After ensuring both of those tubes were fully mixed and then placed in the cooler, the 3 tubes containing the DNA from pine nut, corn mufÏn mix, and soy chips were removed from the bench top cooler and thawed by placing them in your hand. The tubes containing the foods were labeled 1-3 and the positive control was labeled 4. Unlike the positive control which already contains all necessary components to perform PCR, those components needed to be added to the 3 foods. To start, 20 microliters of primer was added using a pipet dispersing the liquid on the side of the tube. For each tube, a new tip was used to prevent cross contamination. Then, 5 microliters of DNA template were added in the same manor in the 3 tubes. Next, 25 microliters of positive control in tuber number 4. Before being placed in the PCR centrifuge all 4 of the tube’s caps were pressed down and tapped on the lab bench to ensure that all material is at the base of the tubes. The process of PCR occurred when the tubes were placed in the Eppendorf flex lid thermal cycler. The GMA preset on the machine runs at 94°C of 5 minutes, 94°C for 60 seconds, 58°C for 60 seconds, and then 72°C for 60 seconds. These steps continued for 2 hours and 20 minutes. Step 3: Preparing Agarose Gel and Running Gel Electrophoresis When PCR is taking place the 2% agarose gel was prepared. 50 milliliters of agarose and Tris borate EDTA buffer was added to a beaker and then microwaved until boiling. After cooled 10 microliters of ethidium bromide is added to the beaker and mixed until all color dissipates. When stirring it was important to be gentle to avoid bubbles which was should up in the final product. After fully mixed the liquid was poured into chamber with a comb and let sit for 20 to 30 minutes. When the PCR process finished, it was then time to run the gel. 25 microliters of each sample were dispersed in to well starting with the ladder in lane 1. The samples will run at 130 volts for approximately 1 hour and then they are ready to be viewed for results under UV light. Results: Figure #1: Lane 1: Ladder Lane 2: Negative control (pine nut)  No bands (error) Lane 3: GMO positive PCR Control  3 bands Lane 4: Corn mufÏn mix (positive control)  3 bands Lane 5: Chips w/ soy  No bands (error) Discussion: Analyze your results in depth, make conclusions regarding your results, explain the support for the conclusion(s) you’ve drawn. Did you accept or reject your hypothesis? Why? The final product of the gel shows some errors in either the extraction of DNA or PCR in some of the samples. Lane 2 and 5 show no bands and the genotype of the sample cannot be determined. The negative control that is in lane 2 would have had the tubulin gene at bp 162 and no bp at 187. Lane 5 contains the soy sample and shows no bands. According to the NonGMO Project, soy is the number one genetically modified crop in the world. If the soy used in the lab was in fact a GMO then it would have had a band at both bp 162 and 187 meaning the gene for tubulin and 35S?NOSt were present. If the soy was not a GMO, then there would only be a band at bs 162. Both the GMO positive PCR control and corn mufÏn mix, in lanes 3 and 4, had bands at bp 162 and 187 meaning they are GMOs. The corn mufÏn mix being positive for being a GMO makes sense because it is a very processed food. I accept my hypothesis because the gel proved that the corn mufÏn mix sample is a GMO.