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The Growth of GM foods• From 1996 to 2003
• Global farmland used to grow transgenic crops increased 40 fold
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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
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GM Maize Products• 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
Corn Borer damage
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The Development of a Unique Protocol for Use in Testing for
Genetically Modified Ingredients in Maize Products
Caitlin Forsyth
W.F. West High School
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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
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Hypothesis
• It was hypothesized that maize products from the Midwest would be genetically modified, and more specifically, with the Cry1A(b) gene
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Methods & Procedures
• Homogenization of test and control foods
• Extraction of DNA• PCR on Extracted DNA• Gel Electrophoresis
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Experimental Design• Multiple Controls
• Positive• Negative• Zein
• Selection of test foods• Midwest• West Coast
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Homogenization of Maize Products
• Coffee Grinder• Mini pestle and mortar
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Extraction of DNA
• Bio-Rad’s InstaGene Matrix
• Machery-Nagel’s Nucleo-Spin Food Extraction Kit
• Qiagen’s Plant Extraction Kit
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Polymerase Chain Reaction•Test and Control Foods
•Zein•CaMV/NOS•Cry1A(b)•Bar
•Primer sequences•Oligo analyzer
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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.
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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
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The Second Trial
• Annealing Temperatures• Primer melting temperatures
• No amplification of zein, bar, or Cry1A(b)
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The Third Trial• PCR Optimization
• MgCl2
• Qiagen’s Q-Solution• BSA
• Amplification of zein, bar, and Cry1A(b) positive controls
• Unable to duplicate results
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Results-Trial 7Tostitos CA & WA corn Great Value
Corn Green Giant
Libby Organic Corn
Organic Tostitos Padrinos
Safeway Taco
Shells
GV Taco Shells
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Gel Arrangement
250
200
150
100
50
ZeinCry1A(b)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
CaMV/NOS
BarZein Cry1A(b)
Negative
Positive
Test food
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Tostitos Tortilla Chips
•Amplification of zein
•Amplication of CaMV/NOS
•Non-Specific Amplification Cry1A(b) and Bar
250
200
150
100
50
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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
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Subsequent Trials• Second Round PCR• Extraction Techniques• HotStarTaq Polymerase
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Results-Trial 13CA & WA
Corn Del Monte Green Giant Great Value Corn
Great Value Taco
Shells
Libby Organic Corn
Organic Tostitos
Padrinos
Safeway Taco
Shells
Tostitos
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Gel Arrangement• Lane 1
• 50 b.p. ladder• Lane 14
•Lambda/HindIII marker• Negative/Positive Controls
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Tostitos Tortilla Chips
•Amplification of 200 base pair band
•Same streaking pattern as Cry1A(b) positive control
250
200
150
100
50
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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
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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
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Correlation• Geographic
Origin • Padrinos—
California & Texas
• Great Value Taco Shells– Arkansas
• Tostitos Tortilla Chips– Texas
• Libby Organic Corn– New York
• Inconclusive correlation
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Conclusion• Purpose changed
• GM testing manual• Became necessary to develop and
test new protocols
• Protocol still not completed
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Limitations
• PCR inhibitors• Starch
• Highly processed food• Positive Controls
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Future Research
• Touchdown PCR• Third Round PCR
• High annealing temperature
• Positive control designed primers
• Different Taq varieties• DNA repair enzyme• More food samples
• Correlation
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Acknowledgements
• Henri Weeks• Dr. Bryony Wiseman• My parents, Norm & Carolee
Forsyth
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Questions?
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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.
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Qiagen’s Q-Solution• Modifies melting behavior of
DNA• Increases specificity,
amplification success
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MgCl2
• Increasing concentration of MgCl2 increases specificity
• Too high of MgCl2 concentration inhibits the polymerase chain reaction
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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
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Components of a PCR Reaction
•The Template DNA•Primers•A Master Mix containing;
•dNTP’s •Buffer•Taq Polymerase
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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
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DNA Extraction• Physically break cell wall• Lysis buffer
• Opens cell
• DNA binds to membrane• Wash with solutions containing
ethanol to wash away impurities
• Elution
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BSA
• Reduces the frequency of primer dimers
• Reduces PCR inhibition
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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.”
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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
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How Does PCR Work?
Chromosome
Cauliflower Mosaic Virus Promoter Sequence – 203 b.p.
Problem – How do we detect the presence of a modified sequence within an organism’s genome?Plant
Cell
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Polymerase Chain Reaction
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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
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Zein Gene• Naturally
occurring maize gene
• Class of protein• Used to coat
paper cups, buttons, adhesives…
• Can replace gum base in chewing gum
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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
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GM testing of milk/animal products
•GM Protein-based testing•GM hormone testing
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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
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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
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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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Why are some genes easier to amplify than others?
•Primer design optimal
•No self-diming or hetero-diming
•Primer concentration
•Annealing temperature
•PCR cycling conditions
•CaMV/NOS primers took 6 months to optimize
•Some regions of DNA have a secondary structure that affects the binding and annealing of the primers
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Primer Length• 1 in 4^(number of bases)
probability that the primers will match to another place
• For example the difference between 20 and 24 bases…
• 4^20
• 1.1x10^12
• 4^24
• 2.81x10^14
• Difference• 2.80x10^14
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Smeared PCR Product
•Too much starting template•Carryover contamination•Enzyme concentration too
high•Too many cycles
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Little or no PCR Product• Pipetting error, missing reagent• PCR cycling conditions not
optimal• Primers degraded• Problems with starting template• Enzyme concentration too low• Insufficient number of cycles• Extension time too short• Primer design not optimal
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Dangers of GM Food• Health effects
• Puztai•Rats, GE potatoes with lectin,
intestinal problems
• Cornell U.•Moths, milkweed with Bt pollen,
death and lack of maturity
• Increased use of chemicals on crops
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Labeling GMOs Worldwide• Europe
• Foods containing over 1% labeled as GMO+
• Food crises in 1990s
• Asia• Similar to Europe• No threshold established• Thailand non-GMO zones
• United States• Foods with less than 5% GMOs may be
labeled as GMO-free, 3 counties in CA have banned production of GM crops
• World Trade Organization• Banning GM crops• Unnecessary obstacle to international
trade
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Methods of Genetically Engineering Food
• Recombinant DNA• Microinjection• Bioballistics• Electro- and Chemical-Poration
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Large Base Pair Non-Specific Amplification
• Common in degraded DNA• As the PCR cycler is hot, small
fragment sizes denature• Fragments start sticking
together• In next annealing, more bands
stick together• Piecing itself back together
randomly• Very large, non-specific DNA
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TouchDown PCR• High annealing temperature
•Highest specificity• Subsequent levels of lower
temperatures•Further amplification
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Why is it so hard to detect GMOs?
•PCR inhibitors•Polysaccharides•Starch
•Denaturation of DNA•Food processing
•GM material present in small amounts
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Oligo Analyzer• Analyzes bases of primers to make
sure they do not• Hairpin• Self-dimer• Hetero-dimer
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Is Amplified Band What I am Looking For?
•Possible it is not •Sequence the band•Compare to established sequence in GenBank
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dNTPs• Too high of dNTP
concentration inhibits PCR• Binds to the Magnesium, no
nucleotides available for extension, making copies