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Today’s PCR: GMO Detection

Today’s PCR: GMO Detection. What are GM crops? Detecting GM crops Elisa, PCR PCR Analysis Sequences detected (PSII, 35S, NOS) Controls (non-GM, GM-positive) Reaction sets. GMO Investigator Kit. What is a GMO?.

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Today’s PCR: GMO Detection

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  1. Today’s PCR: GMO Detection • What are GM crops? • Detecting GM crops • Elisa, PCR • PCR Analysis • Sequences detected (PSII, 35S, NOS) • Controls (non-GM, GM-positive) • Reaction sets

  2. GMO Investigator Kit

  3. What is a GMO? "genetically modified organism (GMO)" means an organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination

  4. Which foods contain GM product? • 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

  5. Which foods contain GM product? Source: USDA ERS http://www.ers.usda.gov/Data/BiotechCrops/

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

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

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

  9. Method for Genetic Modification of Crops • Choose desirable trait • Clone the gene • Engineer the gene • Transform gene into plant • Backcross GM plant into high yield crops

  10. 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

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

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

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

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

  15. 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?

  16. 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

  17. How to test for GMOs • Test for GMOs by PCR: • Grind food • Extract DNA from sample • Test sample DNA for viable plant DNA • Test sample DNA for genetic modifications

  18. Which foods yield viable plant DNA?

  19. Extract DNA from food

  20. 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

  21. GMO Detection: DNA Extraction

  22. GMO Detection: DNA Extraction

  23. Break to Prepare Food DNA • Control food: Mung Bean • Unknown foods: • ?? • ?? • ?? • Each workgroup to prepare two foods.

  24. 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.

  25. How is the GMO DNA detected? CaMV 35S – Sequence for the promoter of 35S transcript of the Cauliflower mosaic virus. Used in transgenics because it functions in every plant cell NOS- Sequence fornopaline synthase terminator from soil bacterium Agrobacterium tumefacians Used in transgenics because it evolved to be recognized in most plants

  26. What are today’s PCR Primers? Plant: Photosystem II GMO: CaMV35s and NOS (draw out on board)

  27. What is needed for PCR? • Template - the DNA to be amplified • Primers - 2 short specific pieces of DNA whose sequence flanksthe 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

  28. Set up PCR reactions

  29. PCR Reactions - Sample Design Layout: 20ul Plant Master Mix (PMM) plus: • a: 20ul positive control DNA (obtain from instructor) • b: 20ul no template control (pure water) • c: 20ul food A undiluted • d: 20ul food A diluted 1:100 • e: 20ul food A diluted 1:10,000 • f: 20ul food B undiluted • g: 20ul food B diluted 1:100 • h: 20ul food B diluted 1:10,000 20ul GMO Master Mix (GMM) plus • a: 20ul positive control DNA (obtain from instructor) • b: 20ul no template control (pure water) • c: 20ul food A undiluted • d: 20ul food A diluted 1:100 • e: 20ul food A diluted 1:10,000 • f: 20ul food B undiluted • g: 20ul food B diluted 1:100 • h: 20ul food B diluted 1:10,000

  30. The PCR Reaction Heat (94oC) to denature DNA strands Cool (59oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 40 cycles

  31. 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

  32. Analysis of Results 1 2 3 4 5 6 7 GMO positive 1: non-GMO food with plant primers 2: non-GMO food with GMO primers 1 2 3 4 5 6 7 3: Test food with plant primers 4: Test food with GMO primers GMO negative 5: GMO positive template with plant primers 6: GMO positive template with GMO primers 7: PCR MW Ruler

  33. Analysis of Results 1: non-GMO food with plant primers 1 2 3 4 5 6 7 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 10ul of Orange Dye into each PCR tube Load 20ul of PCR Reaction + Dye to each lane Marker has loading dye – load 20ul

  34. GMO Detection: PCR set-up Today a slight modification

  35. GMO Detection: PCR set-up

  36. GMO Detection: Electrophoresis

  37. GMO Detection: Electrophoresis

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