GMO Investigator Kit

InstructorSherri Andrews, Ph.D.North Carolina School of the ArtsWinston-Salem, NC

Why teach GMO testing? • Inquiry-based

• Real-world test

• Environmental Science

• Plant Physiology

• Genetics and biotechnology

• Bioinformatics/Data Mining

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

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

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?

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.

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?

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?

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

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

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

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.

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

Extract DNA from food

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

Set up PCR reactions

50 μl

Volumetric Measurements

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

PCR Review

PCR Animation

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

Why have GM crops? • Growing human population

• Loss of farmable land

• Remediation of soil

• Enrich nutrient content

Desirable Traits • Pest Resistance

• Herbicide Tolerance

• Viral Resistance

• Drought Resistance

• Increased Nutritional Value

• Improved Fruit

• Altered Ripening

Opponents argue • Creation of super pests

• Creation of super weeds

• Loss of biodiversity

• Biotechnology companies control agriculture

• Health concerns

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

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

Clone the gene

Ti plasmidori

Bt gene

Bacillus thuringiensis

Delta endotoxin crystal

Ti genes

Engineer the gene

STOP

Antibiotic resistance

Ti plasmidori

Bt gene

Ti genes

GO

Transform gene into plant

Isolate plant cells

Grow undifferentiated callus

Transform cells

Select cells

Redifferentiate callus

Grow transgenic plant

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

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

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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

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

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