GMO Investigator Kit

Is your food genetically modified?

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

GMO Investigator

ProceduresOverview

What is a GMO?

"genetically modified organism (GMO)"

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 a

ll c

rop

pla

nte

d GM corn GM soy

Sources: 1996-1999 Fernandez and McBride, 2000-2004:

USDA, National Agriculture Statistics Service, Acreage.

Soy-based protein drinks/powders

Potato chipsMeatballs and

burgers

containing soy protein

Soy flour

Wheat flourFriesPuffed corn snacksCorn meal

Cereal (egcornflakes)

PopcornFlavored tortilla chips

Corn bread mix

Salad dressingFried corn snacksTortilla chipsFresh papaya

OilVeggie burgersVeggie sausagesFresh corn

Very Difficult / Not Possible

Less ReliableReliableVery Reliable

Which foods yield viable plant DNA?

Why test for GMO’s?

•LegislationLegislationLegislationLegislation

–US: food labeled “GM-Free” <5% GM

–EU: food labeled “GM” if >1% GM

–Japan: food labeled “GM” if >5%

•ExportExportExportExport

•What about unlabeled food?What about unlabeled food?What about unlabeled food?What about unlabeled food?

How to test for GMOs

ELISA:

Test for presence of proteins expressed from genetic modifications

Pro: Quick, cheap, low tech

Con: Crop specific, protein stability

PCR:

Test for presence of inserted foreign DNA

Pro: 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 tumefacians

Used because it evolved to be recognized in most plants

Laboratory Quick Guide

Extract DNA

from food

50 µl

Volumetric

Measurements

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

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

�Forward

Reverse

• Nucleotides - dATP, dCTP, dGTP, dTTP

• Magnesium chloride - enzyme cofactor

• Buffer - maintains pH & contains salt

• Taq DNA polymerase – thermophillicenzyme from hot springs

Polymerase Chain Reaction

PCR Animationhttp://www.bio-rad.com/LifeScience/jobs/2004/04-0522/04-0522_PV92_PCR.html

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

YYgG

YygG

YYggYygg

YYgG x YYgGYYgG

YYgg

YYGgYYGG

1 32 7654

GMO

positive

GMO

negative

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

7: PCR MW Ruler

Analysis of

Results

1 32 7654

GMO Investigator Kit

Lab Extensions •Independent studies

•Data Mining/Bioinformatics for

specific genes

–E.g. Design primers to the cry genes in Bt corn

•Quantitative Real-Time PCR

Trouble shooting

• False PositivesFalse PositivesFalse PositivesFalse Positives

–Contamination-sterile technique; 10% bleach to clean pipette barrels, mortars & pestles, bench tops; barrier tips for all steps.

• False NegativesFalse NegativesFalse NegativesFalse 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