Testing Foods for Introduced Genes

GMO—Detecting Genetically Modified Foods

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-AnimalsAnimal products

have been proposed or produced▫ Pig engineered to

produce omega-3 fatty acids.

▫ Sheep that express antibodies in milk

GMO’s - PlantsGenetically 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 PlantsFlavr 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 NormalMore 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-CornBt 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 ReadyRoundup is the

brand name of a systemic, broad-spectrum 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 CropsIn 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 FarmingUsing 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 waste-fed 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 CloningThe 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 CloningA 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 CloningA plant promoter (P-35S) is

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 tumor-inducing (Ti) plasmid from Agrobacterium tumefaciens.

35S Resistance

5’ aaaaaaaaaaa 3’

Roundup Ready TransformationAgrobacterium

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 ReadyRoundup 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 PCRDNA Microarray Captured PCR-ELISA Quicktest strip

How to Detect a GMOIsolate 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.   

LOOK FOR THE GENETIC

DIFFERENCE??

Real-time PCRScreening Kits

Target genes: CaMV 35S Promotor, Nos teminator , NptII, Bar, FMV promotor, Pat Reference gene: 18S rRNAQuantitative Kits

Roundup Ready soybean Bt176 MaizeEvent-specific detection kits

GTS40-3-2, Bt176, Mon810, Bt11, GA21, T25, RT73

35S PROMOTER INDICATES GMOHerbicide 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 GMOThe 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:   60 C 30”

Extending step:   72 C 30’

34X..35S ----162 base pairsTubulin-187 base

pairs

RG DP BS AS EC AS KR FD CB AM + c TL ASca TJ TF SC DH DH DH

20001600 1000 500

20001600 1000 500

PCR + - - + + + + + + + + + + - + + - + +GMO - - - - - + - - + - - + - + + - -

Results: May 201015 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 WM RP TS AH HB EB CV AL AK M M LH NF BF TW MH SB ZP + MM

S35 Promoter CHIPS Cracker Veg. pepperoni Tor. Chip pretzel PC

Tubulin Control PSII Control PC

500

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.