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GMO Investigator Kit
InstructorSherri Andrews, Ph.D.North Carolina School of the ArtsWinston-Salem, NC
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Why teach GMO testing? • Inquiry-based
• Real-world test
• Environmental Science
• Plant Physiology
• Genetics and biotechnology
• Bioinformatics/Data Mining
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GMO Workshop Time Line • Introduction to GM foods
• DNA extraction of food products
• Set up PCR reactions
• Electrophorese PCR products
• Analysis and interpretation of results
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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
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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?
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Which foods contain GM product?
0
10
20
30
40
50
60
70
80
90
100
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
% o
f al
l cro
p p
lan
ted GM corn GM soy
Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage.
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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
Which foods yield viable plant DNA?
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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?
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How to test for GMOs ELISA:
Test for presence of proteins expressed from genetic modificationsPro: Quick, cheap, low tech
Con: Crop specific, protein stability
PCR:
Test for presence of inserted foreign DNAPro: ID different GM crops, DNA stability
Con: Expensive, timely
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How to test for GMOs
Test for GMOs by PCR:
1. Grind food
2. Extract DNA from sample
3. Test sample DNA for viable plant DNA
4. Test sample DNA for genetic modifications
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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
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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.
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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 for nopaline synthase terminator from soil bacterium Agrobacterium tumefaciansUsed because it evolved to be recognized in most plants
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Extract DNA from food
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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
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Set up PCR reactions
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50 μl
Volumetric Measurements
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The PCR Reaction
What do you need?
What is needed for PCR?
• Template - the DNA to be amplified
• Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence
ForwardReverse
• Nucleotides - dATP, dCTP, dGTP, dTTP
• Magnesium chloride - enzyme cofactor
• Buffer - maintains pH & contains salt
• Taq DNA polymerase – thermophillic enzyme from hot springs
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PCR Review
PCR Animation
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The PCR Reaction
How does it work?
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
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Why have GM crops? • Growing human population
• Loss of farmable land
• Remediation of soil
• Enrich nutrient content
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Desirable Traits • Pest Resistance
• Herbicide Tolerance
• Viral Resistance
• Drought Resistance
• Increased Nutritional Value
• Improved Fruit
• Altered Ripening
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Opponents argue • Creation of super pests
• Creation of super weeds
• Loss of biodiversity
• Biotechnology companies control agriculture
• Health concerns
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Method for Genetic Modification of Crops
1. Choose desirable trait
2. Clone the gene
3. Engineer the gene
4. Transform gene into plant
5. Backcross GM plant into high yield crops
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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
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Clone the gene
Ti plasmidori
Bt gene
Bacillus thuringiensis
Delta endotoxin crystal
Ti genes
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Engineer the gene
STOP
Antibiotic resistance
Ti plasmidori
Bt gene
Ti genes
GO
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Transform gene into plant
Isolate plant cells
Grow undifferentiated callus
Transform cells
Select cells
Redifferentiate callus
Grow transgenic plant
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Backcross GM plant into high yield crops
GM plant = yyGG
High yield plant = YYgg
YYgg x yyGG YyGg
YYgg x YyGg
YYgGYygGYYggYygg
YYgG x YYgG YYgGYYggYYGgYYGG
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1 32 7654
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 32 7654
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GMO Investigator KitLab Extensions
• Independent studies
• Data Mining/Bioinformatics for specific genes
–E.g. Design primers to the cry genes in Bt corn
• Testing for blended foods
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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
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GMO Investigator Kit contents
Not Included but required:•Thermal cycler•Water bath/heat block•Electrophoresis Module (agarose, TAE buffer & Fast Blast DNA stain)•Electrophoresis equipment & power supply•2-20 ul pipettes & barrier tips
• Bio-Rad certified Non-GMO food• InstaGene• Master Mix• GMO primers• Plant PSII primers• GMO & PSII positive control DNA • PCR MW Ruler• DPTPs, microtubes, PCR tubes, foam
floats• Manual