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The Growth of GM foods

The Growth of GM foods. From 1996 to 2003 Global farmland used to grow transgenic crops increased 40 fold . The Genetically Modified Food Debate. Concerned Consumers New toxins and allergens Glyphosate Super Weeds Loss of bio-diversity in crops The disturbance of ecological balance

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The Growth of GM foods

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  1. The Growth of GM foods • From 1996 to 2003 • Global farmland used to grow transgenic crops increased 40 fold

  2. The Genetically Modified Food Debate • Concerned Consumers • New toxins and allergens • Glyphosate • Super Weeds • Loss of bio-diversity in crops • The disturbance of ecological balance • Supporters • Increased yields • Increased nutritional value • Hardy crops • Vaccines • Rapid maturity • More people, fewer resources

  3. GM Maize Products Corn Borer damage • 45% of all maize grown in US is GM • cry1A(b) gene • Pesticide • Syngenta, Monsanto, Pioneer Hi-Bred • Midwestern United States • Bar gene • Herbicide resistance • Bayer, Aventis

  4. The Development of a Unique Protocol for Use in Testing for Genetically Modified Ingredients in Maize Products Caitlin Forsyth W.F. West High School

  5. Purpose • The purpose of this research was to find a correlation between specific genetic modification and geographic origin of maize products, using a specially designed protocol

  6. Hypothesis • It was hypothesized that maize products from the Midwest would be genetically modified, and more specifically, with the Cry1A(b) gene

  7. Methods & Procedures • Homogenization of test and control foods • Extraction of DNA • PCR on Extracted DNA • Gel Electrophoresis

  8. Experimental Design • Multiple Controls • Positive • Negative • Zein • Selection of test foods • Midwest • West Coast

  9. Homogenization of Maize Products • Coffee Grinder • Mini pestle and mortar

  10. Extraction of DNA • Bio-Rad’s InstaGene Matrix • Machery-Nagel’s Nucleo-Spin Food Extraction Kit • Qiagen’s Plant Extraction Kit

  11. Polymerase Chain Reaction • Test and Control Foods • Zein • CaMV/NOS • Cry1A(b) • Bar • Primer sequences • Oligo analyzer

  12. Gel Electrophoresis • The PCR products were run on 2% agarose gels with a 14-tooth comb • Zein • 277 b.p. • CaMV/NOS • 203 b.p. CaMV • 225 b.p. NOS • Bar • 600 b.p. • Cry1A(b) • 240 b.p.

  13. The First Trial • European Joint Research Council GM Testing Protocol • Annealing temperatures • Primer sequences • No amplification of Cry1A(b), bar, or zein • Harder than anticipated

  14. The Second Trial • Annealing Temperatures • Primer melting temperatures • No amplification of zein, bar, or Cry1A(b)

  15. The Third Trial • PCR Optimization • MgCl2 • Qiagen’s Q-Solution • BSA • Amplification of zein, bar, and Cry1A(b) positive controls • Unable to duplicate results

  16. Results-Trial 7 Great Value Corn Tostitos CA & WA corn Green Giant GV Taco Shells Libby Organic Corn Organic Tostitos Safeway Taco Shells Padrinos

  17. Gel Arrangement 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Negative Positive Test food 250 200 150 100 50 CaMV/NOS Cry1A(b) Cry1A(b) Zein Zein Bar

  18. Tostitos Tortilla Chips • Amplification of zein • Amplication of CaMV/NOS • Non-Specific Amplification Cry1A(b) and Bar 250 200 150 100 50

  19. Great Value Taco Shells • No amplification of zein • Amplification of CaMV/NOS • Non-specific amplification of Cry1A(b) and Bar 250 200 150 100 50

  20. Subsequent Trials • Second Round PCR • Extraction Techniques • HotStarTaq Polymerase

  21. Results-Trial 13 CA & WA Corn Del Monte Green Giant Great Value Corn Safeway Taco Shells Great Value Taco Shells Libby Organic Corn Organic Tostitos Padrinos Tostitos

  22. Gel Arrangement • Lane 1 • 50 b.p. ladder • Lane 14 • Lambda/HindIII marker • Negative/Positive Controls

  23. Tostitos Tortilla Chips • Amplification of 200 base pair band • Same streaking pattern as Cry1A(b) positive control 250 200 150 100 50

  24. Green Giant Canned Corn • No amplification of 200 base pair band • No amplification of zein • Non-specific amplification of Cry1A(b) 250 200 150 100 50

  25. Overall Results • CaMV/NOS positive: • Great Value Taco Shells • Libby Organic Corn • Padrinos Restaurant Style Tortilla Chips • Tostitos Tortilla Chips • Nonspecific Amplification • Cry1A(b) • Bar

  26. Correlation • Geographic Origin • Padrinos—California & Texas • Great Value Taco Shells– Arkansas • Tostitos Tortilla Chips– Texas • Libby Organic Corn– New York • Inconclusive correlation

  27. Conclusion • Purpose changed • GM testing manual • Became necessary to develop and test new protocols • Protocol still not completed

  28. Limitations • PCR inhibitors • Starch • Highly processed food • Positive Controls

  29. Future Research • Touchdown PCR • Third Round PCR • High annealing temperature • Positive control designed primers • Different Taq varieties • DNA repair enzyme • More food samples • Correlation

  30. Acknowledgements • Henri Weeks • Dr. Bryony Wiseman • My parents, Norm & Carolee Forsyth

  31. Questions?

  32. Qiagen’s HotStarTaq Polymerase • Qiagen’s HotStarTaq Polymerase was used because it minimizes nonspecific amplification products, primer-dimers, and background. • MasterMix and PCR reactions can be set up at room temperature.

  33. Qiagen’s Q-Solution • Modifies melting behavior of DNA • Increases specificity, amplification success

  34. MgCl2 • Increasing concentration of MgCl2 increases specificity • Too high of MgCl2 concentration inhibits the polymerase chain reaction

  35. The Bar Gene • Transgenic cells and plants expressing this gene are resistant to the herbicides Basta (Europe), Bialaphos (Japan) and Ignite (USA) • Glufosinate ammonium tolerance • Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine • Glufosinate acts enough like glutamate, that it blocks the enzyme's usual activity

  36. Components of a PCR Reaction • The Template DNA • Primers • A Master Mix containing; • dNTP’s • Buffer • Taq Polymerase

  37. What genes are used in GMFs? • Foreign genes are translated into foreign proteins • Desired effects • Pesticides • BT • More desirable size & color, desirable to consumer • Resistant to harsh environmental conditions • More nutritional value, altered vitamin, mineral, and fat contents • Food to vaccinate against diseases • Banana that produces an antigen found in the outer coat of the Hepatitis B virus

  38. DNA Extraction • Physically break cell wall • Lysis buffer • Opens cell • DNA binds to membrane • Wash with solutions containing ethanol to wash away impurities • Elution

  39. BSA • Reduces the frequency of primer dimers • Reduces PCR inhibition

  40. Glyphosate • On the market now, Monsanto • Glyphosate kills tadpoles, decline in frog population • Hardell and Eriksson • link between glyphosate and lymphoma, “link was not statistically significant and was within the realm of random variation.”

  41. Taq Polymerase • Isolated from bacterium that lives in heat vents and hot springs • Commerically sold Taq DNA polymerase (low fidelity) has an error rate of one in 8 million nucleotides • Adds nucleotides to the primer ends • Proofreading mechanism • Vent • Pfu

  42. How Does PCR Work? Chromosome Problem – How do we detect the presence of a modified sequence within an organism’s genome? Plant Cell Cauliflower Mosaic Virus Promoter Sequence – 203 b.p.

  43. Polymerase Chain Reaction

  44. Cauliflower Mosaic Virus Promoter Sequence – 203 b.p. After 30 cycles Millions of copies of the 203 b.p. virus promoter sequence common in many GMO’s

  45. Zein Gene • Naturally occurring maize gene • Class of protein • Used to coat paper cups, buttons, adhesives… • Can replace gum base in chewing gum

  46. Cry1A(b) gene • The cry1Ab gene produces protein Cry1Ab • Cry proteins, of which Cry1Ab is only one, binds to the lining of the gut of lepidopteran insects • Pores are formed that disrupt ion flow, causing gut paralysis and cell lysis and eventual death

  47. GM testing of milk/animal products • GM Protein-based testing • GM hormone testing

  48. Gel Electrophoresis • DNA (sugar-phosphate backbone) negatively charged • Loaded on negative side, attracted to positive side • Gel forms porous matrix • Electric current carried through running buffer • Small fragments go through faster, end of gel • Ethidium bromide stain

  49. Organic Foods • Modified genes were found in normal plants more than 13 miles away from the source • Products labeled "organic" must consist of at least 95 percent organically produced ingredients

  50. Nonspecific PCR Product • PCR cycling conditions not optimal • Annealing temperature too low • Primer concentration not optimal • Primers degraded • Primer design not optimal • More bases in primer design

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