Biology 3210 Laboratory
Lab Report 1
Detection of genetically modified maize in from commonly available corn food product
A genetically modified organism (GMO) is an organism which has had its genome modified by the addition of genetic material from another species (Steynen, 2012). There are many debates surrounding genetically modified organisms on both personal levels, global levels and anywhere in between. There are issues on whether food produced with GMOs is safe, if it is ethical and if and how theses food products should be labeled. There are also several issues involving patent rights and intellectual property, which span the world from Canada to Brazil to India. Monsanto Canada Inc. v. Schmeiser is just one recent example in 2004. Monsanto, an agricultural biotechnology company sued a farmer for patent infringement when refused to pay a licencing fee when the seed he harvested was found to Roundup-resistant. The round-up resistant gene was both developed and patented by Monsanto. The objective of this experiment is to isolate the DNA from commonly available corn (Zea mays Zea saccharata or Zea rugosa,) products and test for evidence of genetic modification. This experiment will proceed in three steps: PCR, isolation and visualization. PCR or polymerase chain reaction was developed by Kary Mullis in 1983 and can amplify one or several strands or sections of DNA to millions or even billions in a matter of hours. PCR involves a DNA sample, primers, polymerases, DNA nucleotides, a buffer and a thermocycler. The primers are complementary region to certain stretches of DNA. They mark the boarders of the DNA region one wishes to amplify and provide a place for a polymerase to bind. The standard DNA polymerase now used in PCR is isolated from Thermus aquaticus. T. aquaticus is found in hot springs and provides a heat stable polymerase that can withstand high temperature PCR conditions. This enzyme has been modified for higher yields and high fidelity. For example, New England Biolabs’ Phusion DNA polymerase has a fidelity 50x that of the standard polymerase and can amplify regions 15kb larger (NEB, 2012). The polymerase, primers, nucleotides and sample are properly mixed into a buffer and positioned into a thermocyler which goes through 3 distinct heat cycles: denaturation, annealing and elongation. Denaturation separates the strands, annealing allows the polymerase to bond to the primer-DNA complex, and elongation allows for the polymerase to add nucleotides and create a complementary strand. When this cycle is repeated over and over it amplifies the region between the primers. The primers (forward and reverse) we will use in this experiment are Ivr1, Cry1, Camv and Nos. The Ivr1 gene codes for maize invertase, an enzyme which catalyzes the hydrolyses the plant disaccharide sucrose into the monosaccharaides glucose and fructose(Xu, 1995). The complimentary 226 bp primer (Steynen, 2012) for this region of DNA can be used to screen for maize genomic DNA. The Cry1 Ab gene is form Bacillus thuringiensis that encodes for a crystalline protein endotoxin. This gene can transformed to Z. mays and the plant can express this protein in its tissue. This protein is can degrades the stomach of butterflies and moth larvae that feed on the plant (Stanley-Horn, 2001). If application with primers complementary to this B. thuringiensis, 184bp gene (Steynen, 2012) are found in corn products, this is an indication of genetic modification. The Camv gene encodes for the CaMV 35S promoter from the cauliflower mosaic virus. The promoter is a very strong promoter, much stronger than the ones found in corn (Cummins, 2000). This 199bp promoter region (Steynen, 2012) is widely used in creation of GMOs genes when one wishes to express at it high levels: the Cry1 Ab for example. The Nos gene encodes for a 127 bp (Steynen, 2012) transcriptional terminator from Agrobacterium tumefaciens (Luo, 2007)....
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