Genetic Modification in Papaya and Fritos® Corn ChipsDocument Transcript
Genetic Modification in Papaya and Fritos® Corn Chips Lester Rosario University of Puerto Rico, Cayey Campus Abstract The purpose of this experiment was to test store-bought food products for thepresence of genetically modified organisms (GMOs). The hypothesis was: Both the papayaand the corn chips are genetically modified organisms (GMOs). In order to test thehypothesis, first, the DNA of papaya and corn chips was extracted. Then, in 4 PCR tubes,20 µL of Plant Molecular Mix (PMM) were added, and in other 4 tubes, 20 µL of GMOMolecular Mix (GMM) were added. Two tubes (one with PMM and other with GMM)contained papaya DNA and other 2 tubes (one with PMM and other with GMM) containedcorn chips DNA. Afterwards, PCR and electrophoresis with agarose gel were carried out tothe samples. According to the data obtained, the papaya and the corn chips didn’t show aband associated with the presence of GMO DNA sequences. However, these results wereunreliable because DNA degradation occurred. Introduction Currently, genetically modified (GM) foods do not have to be labeled as such in theUS and foods with less than 5% genetically modified content can be labeled "GMO-free".In Europe and Asia, genetically modified foods do require labeling if they contain >1% GMcontent. Many people think that the use of GM crops has an effect. They say that there is apossibility to create super-weeds through cross-pollination with herbicide-resistant crops, orthat super-bugs will evolve that are no longer resistant to the toxins in pest-resistant crops.Many are concerned with potential allergic reactions to the novel proteins or antibioticresistance arising from the selectable markers used to develop the crops or other unforeseeneffects on public health. Proponents of genetically modified foods argue these crops areactually better for the environment. Fewer toxic chemicals are put into the environment andthus fewer toxic chemicals can harm the environment and human health. In addition, thesecrops can preserve arable land by reducing stresses on the land, improve the nutritionalvalue of food in developing countries, and allow crops to be grown on previouslyunfarmable land. (Bio-Rad Laboratories, Inc.). However, one of the main difficultieswhich farmers will encounter when growing GM crops is no way to contain pollenmovement. In the case of oilseed rape, researchers have found that its pollen can travel upto 4km and can escape from fields even when they are surrounded by barrier crops toprevent this (Thompson, 2003). This can lead to contamination of other non-GMO crops(Simpson, 2003). The purpose of this experiment is to test store-bought food products for the presenceof genetically modified organisms (GMOs). There are two different GMO-associated DNAsequences: the 35S promoter of the cauliflower mosaic virus (CaMV 35S) and the
terminator of the nopaline synthase (NOS) gene of Agrobacterium tumefaciens. One orboth of these sequences are present in most of the genetically modified crops that areapproved for distribution in North America, Asia, and Europe. The promoter serves as adocking site for RNA polymerase and a signal for where it should start transcribing a gene.The terminator is the signal to stop transcription. The native promoters and terminators ofunmodified genes interact with other components of a host cell to turn genes on or offdepending on cell type and situation, but scientists can engineer the constructs for GMOs sothat the foreign gene is continually transcribed and the foreign protein is producedthroughout the entire plant. On the other hand, most plants contain in their genome a thirdsequence of DNA, the photosystem II chloroplast gene, and this makes it easy to ensurewhether an extraction of DNA is from a plant or not. When using PCR and gelelectrophoresis, the DNA fragments amplified from the 35S promoter and NOS terminatorare 203 and 225 base pairs (bp) respectively, and the fragments of the photosystem II geneis 455 bp. Basing on all this theory, the hypothesis is: Both the papaya and the corn chipsare genetically modified organisms (GMOs). (Bio-Rad Laboratories, Inc.) Methods Extraction of DNA from food samples First of all, using a balance, weigh 2g of papaya and 2g corn ships separately. Then,using a mortar and adding 5 mL of water, grind for 2 minutes the 2g of papaya and 2g ofcorn ships separately. Obtain 50 µL from the slurries of papaya and corn ships and add itrespectively to 2 screwcap tubes containing 500 µL of InstaGen matrix each. Then, shakethe tubes and place them in 95°C water bath for 5 minutes. Later, place the tubes in acentrifuge in a balanced conformation, and centrifuge for 5 minutes at max speed. Finally,the tubes are stored in a refrigerator. Set up PCR reactions Label 8 PCR tubes, going from number 1 to number 8. Afterwards, in the tubes 1,3, 5, and 7, add 20 µL of Plant Molecular Mix (PMM) and in the remaining tubes add 20µL of GMO Molecular Mix (GMM). In the tubes 1 and 2, add 20 µL of non-GMO solution(negative control). In the next two tubes (3 and 4), add 20 µL of GMO positive control.Then, obtain 20 µL from the supernatant of the screwcap tube containing the papaya DNAand add it to the tubes 5 and 6. With the corn ships DNA (inside the other screwcap tube)do the same step, but add the supernatant to the tubes 7 and 8. Finally, place the PCR tubesin the thermal cycler.
Electrophoresis of PCR products First of all, prepare an agarose gel. Then, obtain the PCR tubes from the thermalcycler and add 10 μl of Orange G loading dye to each sample and mix well. Load 20 μL ofthe molecular weight ruler into the lanes 1 and 10, and 20 μL of each sample into the gel asfollows: non-GMO with PMM in lane 2, non-GMO with GMM in lane 3, GMO positivecontrol with PMM in lane 4, GMO positive control with GMM in lane 5, papaya withPMM in lane 6, papaya with GMM in lane 7, corn chips with PMM in lane 8, and cornchips with GMM in lane 9. Run the agarose gel for 30 min at 100 V. Once it is done, takeout the gels and place on them Ethidium Bromide with buffer. Wait for 20 minutes.Finally, remove the Ethidium Bromide and observe the gels. Results Figure 1: Electrophoresis results for GMO and Plant material. Discussion According to the data obtained, in lane 2, where there was the negative controlwhich was known non GMO certified seed with plant primers, a band of 455bp was notpresent. This not necessarily is an error as the plant primer, although most plants carry thegene not all plants do. In this case it is a tested control so we did expected to observe a bandfor the negative plant control. This error could be DNA degradation, wrong pipetting orDNA extraction, etc. In lane 3, where there was the non-GMO food with GMO primers, noband in 203bp appeared as it was expected with some possible degradation. In lane 4,where there was the GMO-positive template with plant primers, a band in 455bp was
observed as expected, confirming plant DNA material. In lane 5, where there was theGMO-positive template with GMO primer, band comprising the 203bp was shownsuccessfully. In lane 6, where there was the papaya with plant primers, it was observed aband of 455bp. This means that the plant DNA of papaya was successfully extracted fromthe sample. In lanes 7 and 9, the samples of papaya and corn chips respectively with GMOprimers, didn’t show neither a band of 203bp nor a band of 225bp. This means that thepapaya and the corn chips aren’t GMOs. However, DNA degradation was also observed,so this experiment should be repeated to obtain more reliable data. Finally, in lane 8, thecorn ships with plant primers showed no significant band. This means that there was againa problem with the sample DNA extraction most probably.ReferencesBio-Rad Laboratories, Inc. Biotechnology Explorer™ GMO Investigator™ KitSimpson, EC et al. 2003. Gene flow in genetically modified herbicide tolerant oilseed rape(Brassica napus) in UK. Gene Flow and Agriculture: Relevance for Transgenic Crops.2003 BCC Symposium Proceedings No. 72 pp 75-81.Thompson, CE et al 2003. Regional patterns of gene flow and its consequence for GMoilseed rape. Gene Flow and Agriculture: Relevance for Transgenic Crops. 2003 BCCSymposium Proceedings No. 72 pp 95-100.