Download GMO—Detecting Genetically Modified Foods

GMO—Detecting Genetically Modified
Foods
Testing Foods for Introduced Genes
GMO’s
• Genetically modified organisms have DNA that has been
modified through genetic engineering
• GM foods were first put on the market in the early 1990s
fueling a revolution in agriculture
 For plants…the gene may come from another plant, or from
another species, or from another kingdom.
 Introduced DNA codes for a protein that gives the GMO an
advantage over the wild type
GMO-Animals
Animal products have been
proposed or produced
▫ Pig engineered to produce
omega-3 fatty acids.
▫ Sheep that express
antibodies in milk
GMO’s - Plants
 Genetically modified plant
products: soybean, corn,
canola, and cotton seed oil.
 Genes encode
 herbicide resistance,
 insect resistance,
 drought tolerance,
 frost tolerance,
 delayed fruit ripening and
other traits.
GMO Plants
Flavr Savr tomato
 the first commercially grown
genetically engineered food
granted a license for human
consumption
 Produced by the Californian
company Calgene 1992
 Sold in 1994, and was only
available for a few years
before production ceased
Flavor-sav
Vs Normal
 More resistant to rotting
and softening by adding an
antisense gene which
interferes with the
production of the enzyme
polygalacturonase (see RNA
interference).
 Softening makes the
tomato more susceptible to
being damaged by fungal
infections.
 Wide Variety of Tomatoes
 Picked BEFORE they are
ripe….still very firm
 Artificially ripen with
ethylene gas
 Easier handling and shelf
life
BT-Corn
 Bt corn is a variant of maize,
genetically altered to express
the bacterial Bt toxin
 Poisonous to insect pests.
 In the case of corn, the pest is
the European Corn Borer
BT-Corn
 A gene from a microorganism
Bacillus thuringiensis inserted
into the corn genome.
 The gene codes for a protein
toxin that forms a crystalline
product…the product is eaten
& perforates the larval digestive
tract.
 The pores allow naturally
occurring enteric bacteria such
as E. coli and Enterobacter to
infect the insect causing death
Round-up Ready
 Roundup is the brand
name of a systemic, broadspectrum herbicide
 Produced by Monsanto
 The active ingredient
glyphosate
Round up Ready
 Monsanto also produces
seeds which grow into
plants genetically
engineered to be tolerant
to glyphosate which are
known as Roundup Ready
crops. The genes contained
in these seeds are patented.
Roundup Ready Crops
 In 1996, genetically modified
Roundup Ready soybeans
resistant to Roundup became
commercially available,
followed by Roundup Ready
corn in 1998
 Current Roundup Ready crops
include soy, maize (corn),
canola, sugar beet, and
cotton, with wheat, and alfalfa
still under development.
No Till Farming
 Using Round-Up
eliminates ALL
plants…except those that
are genetically modified
 No need to till (plow-turn
over) the fields.
 Preserves the top soil
“But plow-based
farming in this
region cultivated
an unexpected
yield: the loss of
fertile topsoil that
literally blew
away in the
winds”
Glyphosate Resistance Found!
 The C4 strain of Agrobacterium
 A species of bacteria that was
found growing in the wastefed column at a factory that
made glyphosate.
 The EPSP synthase enzyme
from this bacterium (C4
EPSP synthase) was almost
completely insensitive to
glyphosate
Agrobacterium tumefaciens
 This bacterium infects plants
and injects DNA from a
plasmid into plant cells
 Injected DNA enters the
nucleus and becomes
incorporated into the plant
chromsomes.
 Under normal circumstances
Agrobacterium tumefaciens
causes gall tumors in plants
Roundup Ready Cloning
 The C4 EPSP bacterial gene
was cloned and inserted into
a bacterial plant vector in
order to prepare for cloning
into plants.
 The Monsanto C4 EPSP
cloning vectors first patented
September 13, 1994
Roundup Ready Cloning
 A plasmid vector that will work
in E. coli
 Needs also characteristics that allow
the plasmid to work in Agrobacterium
tumefaciens.
 Needs a promoter…..to turn on
the gene in plants!
Roundup Ready Cloning
 A plant promoter (P-35S) is
35S
inserted at the 5' end.
 This promoter is the 35S promoter
from cauliflower mosaic virus
(CaMV).
 The 3' end of the gene is modified
by inserting the polyadenylation
site (NOS 3') from the nopaline
synthase gene of the tumorinducing (Ti) plasmid from
Agrobacterium tumefaciens.
5’
Resistance
aaaaaaaaaaa 3’
Roundup Ready Transformation
 Agrobacterium tumefaciens.
infects plants and injects
DNA into plant cells where
it enters the nucleus and
becomes incorporated into
the plant chromsomes.
 The recombinat DNA is
transferred and no tumors
are formed.
Roundup Ready
 Roundup Ready soybean was
the first crop plant produced
by Monsanto.
 Today, 90% of the soybean
crop in the USA consists of
Roundup Ready® plants.
 You can't buy soybean
products that don't come
from genetically modified
plants.
How to make a Genetically Modified Plant
 Isolate gene that direct cells to
make protein of interest
 (From bacteria in the sewers of the
chemical plant making RR)
 Attach the gene to the promoter
that works in plant
 (Califlower mosaic virus 35S)
 Insert the promoter-gene and a
gene for selectable marker into
plant cells
 Agrobacterium tumefaciens
 Allow the genetically altered
cells to grow into plants.
Detection technology of GMOs
 Real-time PCR
 DNA Microarray
 Captured PCR-ELISA
 Quicktest strip
How to Detect a GMO
 Isolate DNA from plant tissue
and food products.
 (PCR) is used to assay for
evidence of
 the 35S promoter that
 drives expression of the
glyphosate resistance gene
and many other plant
transgenes.
Real-time PCR
Screening Kits
Target genes: CaMV 35S Promotor, Nos teminator , NptII, Bar,
FMV promotor, Pat
Reference gene: 18S rRNA
Quantitative Kits
Roundup Ready soybean
Bt176 Maize
Event-specific detection kits
GTS40-3-2, Bt176, Mon810, Bt11, GA21, T25, RT73
35S PROMOTER INDICATES GMO
 Herbicide resistance
correlates with an insertion
allele – the 35S promoter –
that is readily identified by
electrophoresis on an
agarose mini-gel.

 Amplification of tubulin, a
protein found in all plants,
provides evidence of
amplifiable DNA in the
preparation, while tissue
from wild-type and
Roundup Ready® soy
plants are positive controls
for the 35S promoter.
Two PCR reactions are performed for each
plant or food sample.
 One primer set amplifies the
35S promoter from
cauliflower mosaic virus.
 The presence of a 35S
product is diagnostic for the
presence of a transgene.
 The 35S promoter is used to
drive expression of the
glyphosate (Roundup)
resistance gene or Bt gene in
edible crops.
 A second primer set amplifies
a fragment of a tubulin gene
and controls for the presence
of plant template DNA.
 Since the tubulin gene is
found in all plant genomes,
the presence of a tubulin
product indicates amplifiable
DNA in the sample isolated.
 Tubulin is a housekeeping
gene
Results of a GMO Test
PCR to Detect GMO
 The following primer sets
were used in the
experiment:

5'-CCGACAGTGGTCCCAAAGATGGAC-3'
(Forward Primer)

5'-ATATAGAGGAAGGGTCTTGCGAAGG-3'
(Reverse Primer)

5'-GGGATCCACTTCATGCTTTCGTCC-3'
(Forward Primer)

5'-GGGAACCACATCACCACGGTACAT-3'
(Reverse Primer)
 PCR Characteristics
 Denaturing step: 94 C 30’
 Annealing step:
 Extending step:
60 C 30”
72 C
30’
 34X..
 35S ----162 base pairs
 Tubulin-187 base pairs
RG DP BS AS EC AS KR FD
CB AM + c
TL
ASca TJ
TF
SC DH DH DH
2000
1600
1000
500
2000
1600
1000
500
PCR + - GMO -
+ + + + +
- - - - +
+ + +
- - +
+
-
+
-
- + +
+ - +
+
+
-
Results: May 2010
15 Samples were successfully amplified with only 4 products testing + for GMO
FD= Wheat (but we had not wheat products?? S C=
TF =
DH= corn pops
+
-
GMO – PCR Results…….Biorad/Carolina Kits
M SL
AB NB CW CT RH LB RB
S35 Promoter
CHIPS
WM RP TS AH HB EB CV AL AK M
Cracker
Veg. pepperoni
M LH NF BF TW MH SB ZP
Tor. Chip
+ MM
pretzel PC
500
Tubulin Control
PSII Control 
PC
Results:
 14 Food items were tested for
genetic modification using the
S35 promoter from CMV as a
marker
 Tubulin or Photosystem II
used for a negative control
 9 samples had + PCR results
 5 samples showed +results
for the S35 promoter
 Problem cracker and tortilla
chip PCR product wrong
size. Chips and pretzel don’t
have + controls
Conclusions:
 Tortilla chips, pretzels, veggie pepperoni and club crackers appear
to contain food from GMO’s
 Wrong size products, no tubulin make conclusion regarding
pretzel and cracker suspect
 Surprize—wheat products have generally not been reported to
contain this genetic modification. Perhaps they also contain corn
or soy products. Further testing would be necessary to confirm.
 Need to optimize the procedure.