How are genetically modified organisms created?

1. How are genetically modified organisms created? • Bacteria • Yeast • Animals • Worms • Flies • Mammals • Plants

2. Ways of inserting foreign DNA into cells

3. Engineering E. coli to produce a foreign protein

4. Synthesis of cDNA using reverse transcriptase Complementary DNA (cDNA) has no introns, and can be expressed in bacteria to make eukaryotic proteins.

5. Genetic engineering of animals

6. Agrobacterium-mediated transformation

7. 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

8. Bacillus thuringiensis Clone the gene Delta endotoxin crystal Bt gene ori Ti plasmid Ti genes Antibiotic resistance

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

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

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

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

13. 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?

14. Which foods contain GM product?

15. Which foods yield viable plant DNA?

16. 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?

17. 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

18. 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.

19. 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 fornopaline synthase terminator from soil bacterium Agrobacterium tumefacians Used because it evolved to be recognized in most plants

20. Extract DNA from food

21. 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

22. Set up PCR reactions

23. 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 1 2 3 4 5 6 7 Analysis of Results GMO positive 1 2 3 4 5 6 7 GMO negative

24. 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