What is a
GMO?
"genetically modified organism
(GMO)" defines an organism in which
the genetic material has been altered
in a way that does not occur naturally
by mating and/or natural
recombination
Typically plant modifications.
Which foods
contain GM
product?
USDA Approval for GM food crops
•Corn
•Soy
•Papaya
•alphalfa
•Canola
•Potato
•Chicory
•Rice
•Squash
•Sugarbeet
•Tomatoes
Approval does not necessarily mean these
crops are distributed
Database of GM crops: www.ceragmc.org/?action=gm_crop_database
100
% of all crop planted
90
GM corn
GM soy
80
70
60
50
40
30
20
10
0
19
95
19
96
19
97
19
98
19
99
20
00
20
01
20
02
20
03
20
04
Which foods
contain GM
product?
Sources: 1996-1999 Fernandez and McBride, 2000-2004:
USDA, National Agriculture Statistics Service, Acreage.
http://en.wikipedia.org/wiki/Genetically_modified_food
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%
– No requirement to identify food as GMO in
Canada or US.
– Required to note nutritional information
not whether GMO (US and Canada)
• Export
• What about unlabeled food?
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 ---allergy to protein
produced
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 which kills corn borers
•HerbicideTolerance: Round Up
Ready crops
Agrobacterium tumifaciens protein with
resistance to Round Up herbicide
(glyphosate)
Delta endotoxin crystal
Transform
gene into
plant
Isolate plant
cells
Grow
undifferentiated
callus
Transform cells
Select cells
Redifferentiate
callus
Grow
transgenic
plant
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
PCR
Which foods yield viable plant DNA?
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
Soy flour
Meatballs and
burgers
containing soy
protein
Soy-based protein
drinks/powders
Potato chips
Wheat flour
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
Extract DNA
from food
Why these
steps?
Mg++
Mg++
•Grinding food to release DNA
•InstaGene chelates divalent ions (e.g.
Mg2+) necessary for DNA degrading
enzymes (e.g. DNases)
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
•Only 50 μl of food transferred otherwise
InstaGene is overwhelmed (~ 5 mg of
original material)
•Boiling releases DNA from food into the
InstaGene solution
InstaGene
•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 – thermophillic
enzyme from hot springs
The PCR
Reaction
Heat (94oC) to denature DNA strands
How does it
work?
Cool (59oC) to anneal primers to
template
Warm (72oC) to activate Taq
polymerase, which extends primers
and replicates DNA
Repeat 40 cycles
Clone the
gene
Bacillus thuringiensis
Delta endotoxin crystal
Bt gene
Ti plasmid
Ti genes
ori
Engineer
the gene
GO
STOP
Bt gene
Ti plasmid
ori
Ti genes
Antibiotic
resistance
Backcross
GM plant
into high
yield crops
YYgg x yyGG
YyGg
YYgg x YyGg
YYgG
YygG
YYgg
Yygg
GM plant =
yyGG
High yield plant =
YYgg
YYgG x YYgG
YYgG
YYgg
YYGg
YYGG
Analysis of
Results
1
2
3
4
5
6
7
GMO
positive
1: non-GMO food with plant primers
2: non-GMO food with GMO primers
1
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
2
3
4
5
6
7
GMO
Investigator
Kit
Lab
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 • Bio-Rad certified Non-GMO food
• InstaGene
Kit
• Master Mix
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
• GMO primers
• Plant PSII primers
• GMO & PSII positive control DNA
• PCR MW Ruler
• DPTPs, microtubes, PCR tubes, foam
floats
• Manual
Analysis of
Results
1
2
3
4
5
6
7
GMO
positive
1: non-GMO food with plant primers
2: non-GMO food with GMO primers
1
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
2
3
4
5
6
7