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GMO Investigator Kit Is your food genetically modified? GMO Investigator Kit Instructors Sherri Andrews, Ph.D. Curriculum and Training Specialist Bio-Rad Laboratories Essy Levy, M.Sc. Curriculum and Training Specialist Bio-Rad Laboratories Why teach GMO testing? • Inquiry-based • Real-world test • Environmental Science • Plant Physiology • Genetics and biotechnology • Bioinformatics/Data Mining • Standards-based GMO Investigator Kit Advantages • Extract and amplify DNA from different food samples • Perform genuine diagnostic procedures • Use PCR and electrophoresis to find GMO foods • Sufficient materials for 8 student workstations • Complete the activity in three 45 minute lab sessions • Laboratory extensions: Real-Time PCR GMO Workshop Time Line • Introduction to GM foods • DNA extraction of food products • Set up PCR reactions • Electrophorese PCR products • Analysis and interpretation of results GMO Investigator Procedures Overview What is a GMO? "genetically modified organism (GMO)" an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination Which foods contain GM product? US Approval for GM food crops •Corn •Soy •Papaya •Canola •Potato •Chicory •Rice •Squash •Sugarbeet •Tomatoes Approval does not necessarily mean these crops are distributed Database of GM crops: www.agbios.com 100 % of all crop planted 90 GM corn GM soy 80 70 60 50 40 30 20 10 0 19 95 19 96 19 97 19 98 19 99 20 00 20 01 20 02 20 03 20 04 Which foods contain GM product? Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage. Which foods yield viable plant DNA? Very Reliable Reliable Less Reliable Very Difficult / Not Possible Fresh corn Veggie sausages Veggie burgers Oil Fresh papaya Tortilla chips Fried corn snacks Salad dressing Corn bread mix Flavored tortilla chips Popcorn Cereal (eg cornflakes) Corn meal Puffed corn snacks Fries Soy flour Meatballs and burgers containing soy protein Soy-based protein drinks/powders Potato chips Wheat flour Why test for GMO’s? •Legislation – US: food labeled “GM-Free” <5% GM – EU: food labeled “GM” if >1% GM – Japan: food labeled “GM” if >5% •Export •What about unlabeled food? How to test for GMOs ELISA: Test for presence of proteins expressed from genetic modifications Pro: Quick, cheap, low tech Con: Crop specific, protein stability PCR: Test for presence of inserted foreign DNA Pro: ID different GM crops, DNA stability Con: Expensive, timely How to test for GMOs Test for GMOs by PCR: 1. Grind food 2. Extract DNA from sample 3. Test sample DNA for viable plant DNA 4. Test sample DNA for genetic modifications Kit Controls • Bio-Rad certified non-GMO food –Verify PCR is not contaminated • GMO positive control DNA –Verify GMO-negative result is not due to PCR reaction not working properly • Primers to universal plant gene (Photosystem II) –Verify viable DNA was extracted Why amplify a plant gene? To confirm that viable DNA was extracted and that negative GM result isn’t due to a non-viable template. Use highly conserved chloroplast gene from Photosystem II – part of the light reaction of photosynthesis. Why use CaMV 35S and NOS? CaMV 35S – Sequence for the promoter of 35S transcript of the Cauliflower mosaic virus. Used because it functions in every plant cell NOS- Sequence for nopaline synthase terminator from soil bacterium Agrobacterium tumefacians Used because it evolved to be recognized in most plants Laboratory Quick Guide Extract DNA from food Volumetric Measurements 50 μl Why these steps? Mg++ Mg++ •Grinding food to release DNA •InstaGene chelates divalent ions (e.g. Mg2+) necessary for DNA degrading enzymes (e.g. DNases) Mg++ Mg++ Mg++ Mg++ Mg++ Mg++ •Only 50 μl of food transferred otherwise InstaGene is overwhelmed (~ 5 mg of original material) •Boiling releases DNA from food into the InstaGene solution InstaGene •Pellet InstaGene and food debris because InstaGene inhibits PCR reaction (Taq needs Mg++) Set up PCR reactions What is needed for PCR? The PCR Reaction What do you need? • Template - the DNA to be amplified • Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence Forward Reverse • Nucleotides - dATP, dCTP, dGTP, dTTP • Magnesium chloride - enzyme cofactor • Buffer - maintains pH & contains salt • Taq DNA polymerase – thermophillic enzyme from hot springs Polymerase Chain Reaction PCR Animation http://www.bio-rad.com/LifeScience/jobs/2004/04-0522/04-0522_PV92_PCR.html The PCR Reaction How does it work? Heat (94oC) to denature DNA strands Cool (59oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 40 cycles Why have GM crops? • Growing human population • Loss of farmable land • Remediation of soil • Enrich nutrient content Desirable Traits • Pest Resistance • Herbicide Tolerance • Viral Resistance • Drought Resistance • Increased Nutritional Value • Improved Fruit • Altered Ripening Opponents argue • Creation of super pests • Creation of super weeds • Loss of biodiversity • Biotechnology companies control agriculture • Health concerns Method for Genetic Modification of Crops 1. Choose desirable trait 2. Clone the gene 3. Engineer the gene 4. Transform gene into plant 5. Backcross GM plant into high yield crops Choose desirable trait Bacillus thuringiensis •Pest Resistance: Bt crops Bacillus thuringiensis protein is a delta endotoxin kills corn borers •HerbicideTolerance: Round Up Ready crops Agrobacterium tumifaciens protein with resistance to Round Up herbicide (glyphosate) Delta endotoxin crystal Clone the gene Bacillus thuringiensis Delta endotoxin crystal Bt gene Ti plasmid Ti genes ori Engineer the gene GO STOP Bt gene Ti plasmid ori Ti genes Antibiotic resistance Transform gene into plant Isolate plant cells Grow undifferentiated callus Transform cells Select cells Redifferentiate callus Grow transgenic plant Backcross GM plant into high yield crops YYgg x yyGG YyGg YYgg x YyGg YYgG YygG YYgg Yygg GM plant = yyGG High yield plant = YYgg YYgG x YYgG YYgG YYgg YYGg YYGG Analysis of Results 1 2 3 4 5 6 7 GMO positive 1: non-GMO food with plant primers 2: non-GMO food with GMO primers 3. Test food with plant primers 1 4: Test food with GMO primers 5: GMO positive template with plant primers 6: GMO positive template with GMO primers 7: PCR MW Ruler GMO negative 2 3 4 5 6 7 GMO Investigator Kit Lab Extensions • Independent studies • Data Mining/Bioinformatics for specific genes – E.g. Design primers to the cry genes in Bt corn • Quantitative Real-Time PCR Trouble shooting • False Positives – Contamination-sterile technique; 10% bleach to clean pipette barrels, mortars & pestles, bench tops; barrier tips for all steps. • False Negatives – No DNA extracted – Possible food type or possibly primers do not work on that plant species – InstaGene matrix transferred to PCR reactions GMO Investigator Kit Contents Not Included but required: •Thermal cycler •Water bath/heat block •Electrophoresis Module (agarose, TAE buffer & Fast Blast DNA stain) •Electrophoresis equipment & power supply •2-20 ul pipettes & barrier tips • Bio-Rad certified Non-GMO food • InstaGene • Master Mix • GMO primers • Plant PSII primers • GMO & PSII positive control DNA • PCR MW Ruler • DPTPs, microtubes, PCR tubes, foam floats • Manual