Transcript Using a Single-Nucleotide Polymorphism to Predict

GMO Investigator
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
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?
% of all crop planted
100
90
GM corn
GM soy
80
70
60
50
40
30
20
10
19
95
19
96
19
97
19
98
19
99
20
00
20
01
20
02
20
03
20
04
0
Sources: 1996-1999 Fernandez and McBride, 2000-2004:
USDA, National Agriculture Statistics Service, Acreage.
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 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

Bio-Rad certified non-GMO food

Kit
Controls

GMO positive control DNA


Verify PCR is not contaminated
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
Volumetric Measurements
50 μl
•Grinding food to release DNA
Why these steps?
Mg++
•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
Mg++
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
PCR Animation
Polymerase Chain
Reaction
http://www.bio-rad.com/LifeScience/jobs/2004/04-0522/04-0522_PV92_PCR.html
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
 Growing
 Loss
Why have GM
crops?
human population
of farmable land
 Remediation
 Enrich
of soil
nutrient content
Desirable Traits
 Pest
Resistance
 Herbicide
 Viral
Tolerance
Resistance
 Drought
Resistance
 Increased
 Improved
 Altered
Nutritional Value
Fruit
Ripening
• Creation of super pests
• Creation of super weeds
Opponents
argue
 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
ori
Ti genes
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
YYgg x yyGG
YYgg x YyGg
YyGg
YYgG
YygG
YYgg
Yygg
GM plant = yyGG
High yield plant =
YYgg
YYgG x YYgG
YYgG
YYgg
YYGg
YYGG
1
Analysis of Results
2
3
4
5
6
7
GMO positive
1: non-GMO food with plant primers
2: non-GMO food with GMO primers
3. Test food with plant primers
1
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