Transcript Slide 1

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
•
Supporters
• Increased yields
• Increased nutritional value
• Hardy crops
• Vaccines
• Rapid maturity
• More people, fewer resources
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
The Development of a Unique Protocol
for Use in Testing for Genetically
Modified Ingredients in Maize Products
Caitlin Forsyth
W.F. West High School
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
Hypothesis
• It was hypothesized that maize
products from the Midwest would be
genetically modified, and more
specifically, with the Cry1A(b) gene
Methods & Procedures
• Homogenization of test and control
foods
• Extraction of DNA
• PCR on Extracted DNA
• Gel Electrophoresis
Experimental Design
• Multiple Controls
• Positive
• Negative
• Zein
• Selection of test foods
• Midwest
• West Coast
Homogenization of Maize Products
• Coffee Grinder
• Mini pestle and mortar
Extraction of DNA
• Bio-Rad’s
InstaGene Matrix
• Machery-Nagel’s
Nucleo-Spin Food
Extraction Kit
• Qiagen’s Plant
Extraction Kit
Polymerase Chain Reaction
•Test and Control Foods
•Zein
•CaMV/NOS
•Cry1A(b)
•Bar
•Primer sequences
•Oligo analyzer
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.
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
The Second Trial
• Annealing Temperatures
• Primer melting temperatures
• No amplification of zein, bar, or
Cry1A(b)
The Third Trial
• PCR Optimization
• MgCl2
• Qiagen’s Q-Solution
• BSA
• Amplification of zein, bar,
and Cry1A(b) positive
controls
• Unable to duplicate
results
Results-Trial 7
Tostitos
CA & WA corn
Great Value
Corn
Green Giant
GV Taco
Shells
Libby Organic
Corn
Organic
Tostitos
Padrinos
Safeway
Taco Shells
Gel Arrangement
1 2 3 4 5
6 7 8 9 10 11 12 13 14
Negative
Positive
Test food
250
200
Zein
CaMV/NOSCry1A(b)
150
100
Zein
Cry1A(b)
50
Bar
Tostitos Tortilla Chips
•Amplification of zein
•Amplication of
CaMV/NOS
•Non-Specific
Amplification Cry1A(b)
and Bar
250
200
150
100
50
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
Subsequent Trials
• Second Round PCR
• Extraction Techniques
• HotStarTaq Polymerase
CA & WA
Corn
Great Value
Taco Shells
Results-Trial 13
Del Monte
Green Giant
Libby Organic Organic Tostitos
Corn
Great Value
Corn
Padrinos
Safeway
Taco Shells
Tostitos
Gel Arrangement
• Lane 1
• 50 b.p. ladder
• Lane 14
•Lambda/HindIII marker
• Negative/Positive Controls
Tostitos Tortilla Chips
•Amplification of 200
base pair band
•Same streaking pattern
as Cry1A(b) positive
control
250
200
150
100
50
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
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
Correlation
• Geographic
Origin
• Padrinos—
California &
Texas
• Great Value
Taco Shells–
Arkansas
• Tostitos
Tortilla
Chips– Texas
• Libby
Organic
Corn– New
York
• Inconclusive
correlation
Conclusion
• Purpose changed
• GM testing manual
• Became necessary to develop and test new
protocols
• Protocol still not completed
Limitations
• PCR inhibitors
• Starch
• Highly processed food
• Positive Controls
Future Research
• Touchdown PCR
• Third Round PCR
• High annealing temperature
• Positive control designed primers
• Different Taq varieties
• DNA repair enzyme
• More food samples
• Correlation
Acknowledgements
• Henri Weeks
• Dr. Bryony Wiseman
• My parents, Norm & Carolee
Forsyth
Questions?
Qiagen’s HotStarTaq Polymerase
•
•
Qiagen’s HotStarTaq
Polymerase was used because it
minimizes nonspecific
amplification products, primerdimers, and background.
MasterMix and PCR reactions
can be set up at room
temperature.
Qiagen’s Q-Solution
• Modifies melting behavior of DNA
• Increases specificity, amplification
success
MgCl2
• Increasing concentration of MgCl2
increases specificity
• Too high of MgCl2 concentration
inhibits the polymerase chain reaction
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
Components of a PCR Reaction
• The Template DNA
• Primers
• A Master Mix containing;
•dNTP’s
•Buffer
•Taq Polymerase
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
DNA Extraction
• Physically break cell wall
• Lysis buffer
• Opens cell
• DNA binds to membrane
• Wash with solutions containing
ethanol to wash away impurities
• Elution
BSA
• Reduces the frequency of primer
dimers
• Reduces PCR inhibition
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.”
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
How Does PCR Work?
Chromosome
Plant Cell
Problem – How do we detect
the presence of a modified
sequence within an
organism’s genome?
Cauliflower Mosaic Virus Promoter Sequence – 203 b.p.
Polymerase Chain Reaction
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
Zein Gene
• Naturally occurring
maize gene
• Class of protein
• Used to coat paper
cups, buttons,
adhesives…
• Can replace gum base
in chewing gum
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
GM testing of milk/animal
products
•GM Protein-based testing
•GM hormone testing
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
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
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
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
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
Smeared PCR Product
• Too much starting template
• Carryover contamination
• Enzyme concentration too high
• Too many cycles
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
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
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
Methods of Genetically Engineering Food
• Recombinant DNA
• Microinjection
• Bioballistics
• Electro- and Chemical-Poration
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
TouchDown PCR
• High annealing temperature
•Highest specificity
• Subsequent levels of lower
temperatures
•Further amplification
Why is it so hard to detect GMOs?
• PCR inhibitors
•Polysaccharides
•Starch
• Denaturation of DNA
•Food processing
• GM material present in small
amounts
Oligo Analyzer
• Analyzes bases of primers to make
sure they do not
• Hairpin
• Self-dimer
• Hetero-dimer
Is Amplified Band What I am Looking For?
•Possible it is not
•Sequence the band
•Compare to established
sequence in GenBank
dNTPs
• Too high of dNTP concentration
inhibits PCR
• Binds to the Magnesium, no
nucleotides available for extension,
making copies