GMO Investigator Kit Is your food genetically modified?

GMO Investigator Procedures Overview

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

Which foods contain GM product?

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?

100 90 80 70 60 50 40 30 20 10 0 GM corn GM soy 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage.

Which foods yield viable plant DNA?

Very Reliable Reliable

Fresh corn Fresh papaya Corn bread mix Corn meal Soy flour Veggie sausages

Less Reliable Very Difficult / Not Possible

Veggie burgers Tortilla chips Flavored tortilla chips Fried corn snacks Popcorn Puffed corn snacks Fries Meatballs and burgers containing soy protein Soy-based protein drinks/powders Potato chips Oil Salad dressing Cereal (eg cornflakes) Wheat flour

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 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

Ca

uliflower

m

osaic

v

irus. Used because it functions in every plant cell

NOS-

Sequence for

no

paline

s

ynthase terminator from soil bacterium

Agrobacterium tumefacians

Used because it evolved to be recognized in most plants

Laboratory Quick Guide

Extract DNA from food

Mg ++

Why these steps?

Mg ++ Mg ++ InstaGene Mg ++ Mg ++ Mg ++ Mg ++ Mg ++ •Grinding food to release DNA •InstaGene chelates divalent ions (e.g. Mg 2+ ) 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 ++ )

Set up PCR reactions

What is needed for PCR?

The PCR Reaction What do you need?

•

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 –

enzyme from hot springs thermophillic

Polymerase Chain Reaction PCR Animation

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

The PCR Reaction How does it work?

Heat ( 94 o C ) to denature DNA strands Cool ( 59 o C ) to anneal primers to template Warm ( 72 o C ) to activate polymerase, which extends primers and replicates DNA Taq 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

Bacillus thuringiensis

•

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)

Delta endotoxin crystal

Clone the gene

Bacillus thuringiensis

Delta endotoxin crystal

Bt gene Ti plasmid Ti genes ori

Engineer the gene

GO STOP Bt gene Ti plasmid Ti genes ori Antibiotic resistance

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 =

yy GG

High yield plant =

YY gg YY gg x yy GG Y y G g YY gg x Y y G g YY g G x YY g G YY g G Y yg G YY gg Y ygg YY g G YY gg YYG g YYGG

Analysis of Results

GMO positive 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 GMO negative 1 2 3 4 5 6 7 1 2 3 4 5 6 7

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