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 7Tostitos CA & WA corn Great Value

Corn Green Giant

Libby Organic Corn

Organic Tostitos Padrinos

Safeway Taco

Shells

GV Taco Shells

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

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

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

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, primer-dimers, 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

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

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