The Growth of GM foods

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

• 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-Rads InstaGene Matrix
• Machery-Nagels Nucleo-Spin Food Extraction Kit
• Qiagens 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
• Qiagens 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)
Zein
Cry1A(b)
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
• PadrinosCalifornia 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
Qiagens HotStarTaq Polymerase

• Qiagens 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
Qiagens 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
• dNTPs
• 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 organisms 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 GMOs
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

51
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

52
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
• 420
• 1.1x1012
• 424
• 2.81x1014
• Difference
• 2.80x1014

53
Smeared PCR Product

• Too much starting template
• Carryover contamination
• Enzyme concentration too high
• Too many cycles

54
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

55
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

56
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

57
Methods of Genetically Engineering Food

• Recombinant DNA
• Microinjection
• Bioballistics
• Electro- and Chemical-Poration

58
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

59
TouchDown PCR

• High annealing temperature
• Highest specificity
• Subsequent levels of lower temperatures
• Further amplification

60
Why is it so hard to detect GMOs?

• PCR inhibitors
• Polysaccharides
• Starch
• Denaturation of DNA
• Food processing
• GM material present in small amounts

61
Oligo Analyzer

• Analyzes bases of primers to make sure they do
  not
• Hairpin
• Self-dimer
• Hetero-dimer

62
Is Amplified Band What I am Looking For?

• Possible it is not
• Sequence the band
• Compare to established sequence in GenBank

63
dNTPs

• Too high of dNTP concentration inhibits PCR
• Binds to the Magnesium, no nucleotides available
  for extension, making copies