This document provides an overview of using a GMO Investigator Kit to test for genetically modified organisms in food. It discusses why GMO testing is taught, outlines the workflow which includes DNA extraction from food samples and PCR amplification to detect genetic modifications. Key points are that primers target conserved plant genes to confirm viable DNA, and transgene sequences like CaMV 35S and NOS to identify specific GMOs. The kit is designed for an inquiry-based lab and includes controls while troubleshooting tips address potential issues.

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

8. 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

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

13. 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.

14. Why use CaMV 35S and NOS ? CaMV 35S – Sequence for the promoter of 35S transcript of the Ca uliflower m osaic v irus. Used because it functions in every plant cell NOS- Sequence for no paline s ynthase terminator from soil bacterium Agrobacterium tumefacians Used because it evolved to be recognized in most plants

15. Extract DNA from food

17. Set up PCR reactions

18. Volumetric Measurements 50 μ l

20. PCR Review PCR Animation

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

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

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

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

30. Backcross GM plant into high yield crops GM plant = yy GG High yield plant = YY gg YY gg x yy GG Y y G g YY gg x Y y G g YY g G Y yg G YY gg Y ygg YY g G x YY g G YY g G YY gg YYG g YYGG

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