Presence of Genetically Modified Organism genes in Carica papaya, Glycine max, Triticum spp. and Zea mays fruits<br />Carlos Santos-Pérez and Valeria Rivera-Torres<br />Department of Biology, University of Puerto Rico, Cayey Puerto Rico<br />_____________________________________________________________________________________________________________________<br />Abstract<br />GMO stands for genetically modified organisms. These have been introduced, in the form of plasmids, an alteration to their DNA sequence. This genetic material includes information that will encode for proteins that can give certain advantages to the organism. PCR tests can detect 85% of all genetically modified crops. This is due to the small number of regulatory genes use to control gene expression. This research focuses on a linear procedure composed by: DNA extraction, Polymerase Chain Reaction and Electrophoresis Gel. The most common are the ones used in this experiment, 35S promoter and NOS terminator. Certainly these results confirmed that the extraction was from a plant since a band was present at 455bp. A smear was present where the band at 203 was supposed to be seen, that indicates the organisms are genetically modified.<br />Introduction<br />Genetically Modified Organism (GMO) refers to animals, plants and microorganisms that because an insertion of a foreign gene or genes, partially or completely change their metabolic pathway. This action causes GMO to synthesize different proteins that also change the traits expressed in the organism. The Genetic Modification in plants can improve agriculture by causing: herbicide, virus and insect resistance of the plant, amplification of the nutritional values of the crop, and better adaption of the plant to different stress exposures. Worldwide, there are four countries which are considered leaders in GMO production: USA, Argentina, Canada and China. Furthermore, the increase of production of GM plants throughout the world can be sustained by establishing that in 1996 there were 1.7 million hectares of GMO’s crops but now the world have 44.2 million hectares approximately. In fact, since 1996 the United States of America are genetically modifying plants. To exemplify this, previous studies established that 90% of the Soy that the people consume at this day is product of genetically modified plant named Glycine max. On the other hand in case of papaya research in that realm of science reveals that only 10% is product of a genetically modified plant named Carica papaya. In case of Zea mays, indicate that 80% of it are products of GMO and Triticums spp. only the 10% is a GM fruit. (Terzi et al 2007)<br />A GM plant contains a promoter and a terminator in order to create the modification in the DNA sequence of the organism. The promoter that is usually use in GM plants in Europe, USA and Asia, is the: 35S promoter of the cauliflower mosaic virus (CaMV 35S); the terminator is usually from the Nopaline Synthase (NOS) gene of Agrobacterium tumefaciens. It can be present only one of them (simplex) or both of them (Duplex). (Scipioni et al. 2008) Using this information it can be identify if a fruit is a GMO or not. In fact, today there is a debate about the commercial identification of these products. In the United States of America there is no regulation about labeling the GM plants product but some organizations want producers to identified if the product is genetically modified or not. A possible solution to this could be the use of ELISA or PCR in order to identify if the fruit is product of a GM plant. The use of ELISA represents a problem. ELISA is an antibody-based test and because of that ELISA can only tests fresh food. This is because of the problem that represents the fast degradation of the protein. In addition, ELISA has to be individualized to every single crop. That is the reason of why PCR is the preferred technique to identify GM plants. The objective is to extract the DNA of papaya, soy, whole corn and wheat and determine whether soy, papaya, corn or wheat is genetically modified products. Because of the literature research it can be expected that papaya and wheat will not be product of a GM plant while soy and corn can be a product of a GM plant. (BioRad® manual, 2010) <br />Material and Methods<br />Introduction<br />The samples were divided into two researchers. One of the researchers worked with Carica papaya and Glycine Max while the other worked with Zea mays and Trititcum spp. <br />Extraction of DNA<br />
Screw cap tubes were labeled one “non GMO” and another one “test”. We added 2g of the certified non- GMO food and put it into the mortar. We added 5ml of distilled water as needed to make a consistent liquid substance and grinded with pestle for at least 2 min to form a slurry. Afterwards, 5ml of water was added to make it smooth enough to pipet. Fifty microliters of ground slurry was added to the screwcap tube containing 500 microliters of InstaGene labeled “non-GMO”. These steps were repeated with test food samples. Fifty microliters of ground test food slurry to the screwcap tube labeled “test”. We flicked the non –GMO food and test food InstaGene tubes and place tubes in a hot plate for 5 minutes. Later these tubes were placed in a centrifuge (Eppendorf Microcentrifuge) for 5 minutes at max speed (13,000rpm). Tubes were stored in freezer until we were ready to conduct PCR. This was made with Carica Papaya, Glycine Max, Triticum spp. and Zea mays.
Polymerase Chain Reaction<br />Each investigator used their samples and conducted PCR. In order to amplify the DNA, the DNA extraction of the respective samples it had to be exposed to PCR. Eight Eppendorf tubes were used to place the control samples and the experimental samples. Also it was used two Eppendorf tubes for the Master-mixes. These two Master-mixes had all the principal ingredients to amplify and replicate the DNA. It had distilled water, Taq Polymerase, one of two primers that are: Green Molecular Mark and Red Molecular Mark. Also these two Master-mixes had Nitrogen Bases, 10X PCR Buffer and Magnesium Chloride. It was used two different primers because the Plant Molecular Marker primer defined if the product it was really a plant. This marker identifies and replicates a gene of the chloroplast specifically in the Photosystem II of the plant. On the other hand, the Genetically Modified Molecular Marker primer identifies and replicates the promoter CaMV 35S and/or the terminator NOS (Red Primer). <br />PCR material was set up as follows:<br />
Materials were kept on ice during the PCR preparation. We added 20 µl of the indicated master mix to each PCR tube, cap tubes. Later 20 µl of the indicated DNA was added to each PCR tube. Avoid InstaGene® pellet at the bottom of the tubes. Place PCR tubes in thermal cycler.<br />This thermal PCR cycler exposed the samples to three steps: denaturing, annealing and elongation. In the step of denaturing the samples were exposed to a temperature of 94˚C in order to separate the two strands of the DNA. Then the temperature cool down to 59˚C and at this moment is when the primer began to stick to the specific places of the DNA in order to initiate the replication. Next the temperature raised to 74˚C and at this temperature was when the Taq polymerase began to stick the nitrogenous. It was done 40 cycles after the initial denaturation but before the final extension. Approximately this part of the experiments lasted 4 hours. <br />Electrophoresis <br />For gel electrophoresis, a 3% Agarose gel was used. This gel was made by first mixing 7.5g of Agarose powder to 250mL of 1% TAE buffer. This solution was mixed until the Agarose dissolved completely. The solution was then heated in microwave oven in intervals of 1 minute, where the solution is heated then stirred. This procedure was repeated until bubbles start to from the Erlenmeyer flask. Once the bubbles form, the solution was covered and left to cool. Note: the solution prepared had some undissolved clumps and was stirred to dissolve it. After the clump was dissolved, the solution was poured unto a casting tray. With a clean pipette tip, bubbles that could affect the results were moved away from the center to the sides. The casting tray was wrapped in plastic and then placed in the refrigerator for one day. <br />The next day involved the insertion into the wells of the gel. First, the gel casting tray is assembled unto the electrophoresis chamber. The eight DNA samples that were amplified were added 10µL of Orange Loading dye. The Loading dye gives the samples a little more weight, so the samples don’t float away. Before dispensing the samples to the gel, 1% TAE buffer was deposited unto the electrophoresis chamber until the point where the buffer was level to the gel. For the electrophoresis, not only did we put 8 DNA samples, but also 2 rulers to measure the bands. Using an eppendorf micropipette, 20µL of each sample was inserted into their corresponding well in the gel. After having depositing each of the samples, the electrophoresis chamber closed and the electrodes are connected. The gel ran for 30 minutes on a constant voltage of 100V. Gel ran for 30 minutes at 100 V. Afterwards it was stained with Ethidium Bromide and visualized using UV light.<br />Results<br />The results of this research can be simplified in an electrophoresis gel. <br />Figure 1: This gel is for Triticum spp. and Zea mays. We were supposed to see the bands of a ladder focusing on 455bp for plant molecular marker and 203 bp for the GMOs.<br />Figure 2: This gel is for Carica papaya and Glycine max. We were supposed to see the bands of a ladder focusing on 455bp for plant molecular marker and 203 bp for the GMOs.<br />Discussion <br />We identify obvious degradation of DNA material. For this reason the entire experiment didn’t prove or revoked our hypothesis. Human errors are involved and affected the results of the investigation. Pipetting, concentrated DNA, lots of primer, evaporation, and contamination are among the possible errors that occurred during this experiment.<br />References<br />Cantamutto, M., & Poverene, M. (2007). Genetically modified sunflower release: Opportunities and risks. Fields Crops Research, 101, 133-134.<br />Jiao, Z., Deng, J., Li, G., & Cai, Z. (2010). Study on the compositional differences between transgenic and non-transgenic papaya(Carica papaya L.). Journal of Food Composition and Analysis, 23, 640-647.<br />Scipioni, A., Saccarola, G., Arena, F., & Alberto, S. (2008). Strategies to assure the abscence of GMO in food products application process in a confectionery firm. Food Control, 16, 569-578.<br />Terzi, V., Malnati, M., Barbanera, M., Stanca, A., & Faccioli, P. (2003). Development of analytical systems based on real-time PCR for Trititcum species- specific detection and quatitation of bread wheat contamination in semolina and pasta. Journal of Cereal Science, 38, 87-94.<br />BioRad® Manual of: Biotechnology Explorer™ GMO Investigator™ Kit<br />