1. GMO Investigator Kit Is your food genetically modified?

2. 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

3. 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?

4. Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage.

5. Very ReliableReliableLess ReliableVery Difficult / Not Possible Fresh cornVeggie sausagesVeggie burgersOil Fresh papayaTortilla chipsFried corn snacksSalad dressing Corn bread mixFlavored tortilla chips PopcornCereal (eg cornflakes) Corn mealPuffed corn snacksFriesWheat flour Soy flourMeatballs and burgers containing soy protein Potato chips Soy-based protein drinks/powders Which foods yield viable plant DNA?

6. 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

7. 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.

8. 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

9. Extract DNA from food

10. Why these steps? Grinding food to release DNA InstaGene chelates divalent ions (e.g. Mg 2+ ) 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 ++ InstaGene

11. Set up PCR reactions

12. The PCR Reaction What 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

13. Polymerase Chain Reaction PCR Animation http://www.bio-rad.com/LifeScience/jobs/2004/04-0522/04-0522_PV92_PCR.html

14. The PCR Reaction How does it work? 1.Heat (94 o C) to denature DNA strands 2.Cool (59 o C) to anneal primers to template 3.Warm (72 o C) to activate Taq polymerase, which extends primers and replicates DNA 4.Repeat 40 cycles

15. Why do you think we have GMO foods?

16. Why have GM crops? Growing human population Loss of farmable land Remediation of soil Enrich nutrient content

17. What are some desirable traits for a GMO to have?

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

19. What are some Arguments against GMO’s?

20. Opponents argue Creation of super pests Creation of super weeds Loss of biodiversity Biotechnology companies control agriculture Health concerns

21. 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

22. 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

23. Clone the gene Ti plasmid ori Bt gene Bacillus thuringiensis Delta endotoxin crystal Ti genes

24. Engineer the gene STOP Antibiotic resistance Ti plasmid ori Bt gene Ti genes GO

25. Transform gene into plant Isolate plant cells Grow undifferentiated callus Transform cells Select cells Redifferentiate callus Grow transgenic plant

26. Backcross GM plant into high yield crops GM plant = yyGG High yield plant = YYgg YYgg x yyGGYyGgYyGg YYgg x YyGg YYgG YygG YYgg Yygg YYgG x YYgG YYgG YYgg YYGg YYGG

27. 1327654 GMO positive GMO negative 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 7: PCR MW Ruler Analysis of Results 1327654

28. 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