GMO Investigator Kit:GMO Investigator Kit Instructor Sherri Andrews, Ph.D. North Carolina School of the Arts Winston-Salem, NC


Why teach GMO testing?:Why teach GMO testing? Inquiry-based Real-world test Environmental Science Plant Physiology Genetics and biotechnology Bioinformatics/Data Mining


GMO Workshop Time Line :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


What is a GMO?:What is a GMO? "genetically modified organism (GMO)" means 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 Which foods contain GM product?


Which foods contain GM product?:Which foods contain GM product? Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage.


Slide 8:Which foods yield viable plant DNA?


Why test for GMO’s?:Why test for GMO’s? Legislation US: food labeled “GM-Free” 1% GM Japan: food labeled “GM” if >5% Export What about unlabeled food?


Slide 10: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


Slide 11:How to test for GMOs Test for GMOs by PCR: Grind food Extract DNA from sample Test sample DNA for viable plant DNA Test sample DNA for genetic modifications


Kit Controls: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?: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?: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


Slide 15:Extract DNA from food


Slide 16:Why these steps? Grinding food to release DNA InstaGene chelates divalent ions (e.g. Mg2+) necessary for DNA degrading enzymes (e.g. DNases) Only 50 μl of food transferred otherwise InstaGene is overwhelmed (~ 5 mg of original material) Boiling releases DNA from food into the InstaGene solution Pellet InstaGene and food debris because InstaGene inhibits PCR reaction (Taq needs Mg++) Mg++ Mg++ Mg++ Mg++ Mg++ Mg++ Mg++ Mg++ InstaGene


Slide 17:Set up PCR reactions


Slide 18:Volumetric Measurements


The PCR ReactionWhat do you need?:The PCR ReactionWhat do you need? What is needed for PCR? 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


PCR Review:PCR Review PCR Animation


Slide 21:The PCR ReactionHow 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? :Why have GM crops? Growing human population Loss of farmable land Remediation of soil Enrich nutrient content


Desirable Traits:Desirable Traits Pest Resistance Herbicide Tolerance Viral Resistance Drought Resistance Increased Nutritional Value Improved Fruit Altered Ripening


Opponents argue :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 :Method for Genetic Modification of Crops Choose desirable trait Clone the gene Engineer the gene Transform gene into plant Backcross GM plant into high yield crops


Slide 26:Choose desirable trait 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) Bacillus thuringiensis Delta endotoxin crystal


Slide 27:Clone the gene Ti plasmid ori Bt gene Bacillus thuringiensis Delta endotoxin crystal Ti genes


Slide 28:Engineer the gene Antibiotic resistance Ti plasmid ori Bt gene Ti genes


Slide 29:Transform gene into plant Isolate plant cells Grow undifferentiated callus Transform cells Select cells Redifferentiate callus Grow transgenic plant


Slide 30:Backcross GM plant into high yield crops GM plant = yyGG High yield plant = YYgg YYgg x yyGG YyGg YYgg x YyGg YYgG YygG YYgg Yygg YYgG x YYgG YYgG YYgg YYGg YYGG


Slide 31:1 3 2 7 6 5 4 GMO positive GMO negative 7: PCR MW Ruler 1: non-GMO food with plant primers 2: non-GMO food with GMO primers 3: Test food with plant primers 4: Test food with GMO primers 5: GMO positive template with plant primers 6: GMO positive template with GMO primers Analysis of Results 1 3 2 7 6 5 4


GMO Investigator KitLab Extensions:GMO Investigator KitLab Extensions Independent studies Data Mining/Bioinformatics for specific genes E.g. Design primers to the cry genes in Bt corn Testing for blended foods


Trouble shooting: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: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