1. Is your Food Genetically Modified?
GMO Investigator
Is your Food Genetically Modified?

2. The Great GMO Debate

3. How Prevalent are GMOs? Corn Soy Papaya US Approval for GM food crops
Canola
Potato
Chicory
Rice
Squash
Sugarbeet
Tomatoes
Approval does not necessarily mean these crops are distributed
Database of GM crops:
Sources: Fernandez and McBride, : USDA, National Agriculture Statistics Service, Acreage.

4. Pros and Cons Advocates Argue Opponents Argue
Growing human population
Loss of farmable land
Remediation of soil
Desirable Traits:
Enrich nutrient content
Pest Resistance
Herbicide Tolerance
Viral Resistance
Drought Resistance
Improved Fruit
Altered Ripening
• Creation of super pests
• Creation of super weeds
Loss of biodiversity
Biotechnology companies control agriculture
Health concerns

5. What is Bt? Is it safe?

6. What are the two ways to test for GMO foods?
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
ELISA used only for fresh food (cheaper, faster). Photosystem II gene that all plants have: use as test for viable plant DNA.
There is also ELISA test for GMO.

7. Our Procedure

8. Day 1 – Make Gels
Done!

9. Day 2 – Prepare Samples

10. What is the plant primer you are testing?
32 kDa psbA gene
D1 Protein
Used because it functions in every plant cell

11. What is the GMO primer you are testing?
CaMV 35S – Sequence for the promoter of 35S transcript of the Cauliflower mosaic virus.
About 65% of food crops use 35S promoter, by adding NOS detection, can detect about 80% of GM foods.

12. 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
Wheat flour
Soy flour
Meatballs and burgers containing soy protein
Potato chips
Soy-based protein drinks/powders
You can search which foods/students projects/ will contain GM. BioRad scientists have already figured out which foods yield better results
DNA is very very stable… Jurassic park DNA 65M years, think about the torture a dorito Corn chip has gone through and you cans still get DNA out of it!!!!

13. Why These Steps? Grinding food releases 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++
InstaGene

14. Day 2 – Run PCR

15. How does PCR work? (3 phases)
Heat (94oC) to denature DNA strands
Cool (59oC) to anneal primers to template
Warm (72oC) to extend primers and replicate DNA
Repeat 40 cycles

16. Which enzyme is important in PCR?
Taq Polymerase
Thermus aquaticus – first discovered at Yellowstone National Park

17. What is needed for PCR? (this is in the master mix)
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

18. Day 3 – Run Gels and Analyze Results