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					       GMO Investigator Kit
Is your food genetically modified?
GMO
Investigator Kit   Sherri Andrews, Ph.D.
                    Curriculum and Training Specialist
Instructors         Bio-Rad Laboratories

                   Essy Levy, M.Sc.
                    Curriculum and Training Specialist
                    Bio-Rad Laboratories
Why teach
GMO testing?   • Inquiry-based

               • Real-world test

               • Environmental Science

               • Plant Physiology

               • Genetics and biotechnology

               • Bioinformatics/Data Mining

               • Standards-based
GMO
Investigator     • Extract and amplify DNA from different food
Kit Advantages     samples

                 • Perform genuine diagnostic procedures

                 • Use PCR and electrophoresis to find GMO
                   foods

                 • Sufficient materials for 8 student
                   workstations

                 • Complete the activity in three 45 minute lab
                   sessions

                 • Laboratory extensions: Real-Time PCR
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   US Approval for GM food crops
contain GM    •Corn
product?
              •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
                                      100

product?
                                       90   GM corn   GM soy




              % of all crop planted
                                       80
                                       70
                                       60
                                       50
                                       40
                                       30
                                       20
                                       10
                                        0
                                        95

                                        96

                                        97

                                        98

                                        99

                                        00

                                        01

                                        02

                                        03

                                        04
                                      19

                                      19

                                      19

                                      19

                                      19

                                      20

                                      20

                                      20

                                      20

                                      20
              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   Popcorn             Cereal (eg
                   chips                                  cornflakes)
Corn meal        Puffed corn snacks Fries                Wheat flour
Soy flour        Meatballs and       Potato chips
                  burgers
                  containing soy
                  protein
                 Soy-based protein
                  drinks/powders
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   ELISA:
for GMOs
              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   Test for GMOs by PCR:
for GMOs
              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   To confirm that viable DNA was
plant gene?     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 – Sequence for the
CaMV 35S   promoter of 35S transcript of the
and NOS?   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
   Why these                          •Grinding food to release DNA
   steps?
                                      •InstaGene chelates divalent ions (e.g.
                                      Mg2+) necessary for DNA degrading
  Mg++
              Mg++                    enzymes (e.g. DNases)
                        Mg++

                            Mg++      •Only 50 μl of food transferred otherwise
                                      InstaGene is overwhelmed (~ 5 mg of
Mg++                                  original material)
                               Mg++
       Mg++
                     Mg++             •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      • Template - the DNA to be amplified
What do you   • Primers - 2 short specific pieces of DNA whose
need?          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
Polymerase
Chain Reaction

                               PCR Animation
                 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
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     1. Choose desirable trait
Genetic
Modification   2. Clone the gene
of Crops
               3. Engineer the gene

               4. Transform gene into plant

               5. Backcross GM plant into high
                  yield crops
Choose
desirable                    •Pest Resistance: Bt crops
trait                           Bacillus thuringiensis protein is a delta
                                endotoxin kills corn borers
  Bacillus thuringiensis

                             •HerbicideTolerance: Round Up
                             Ready crops
                                Agrobacterium tumifaciens protein with
                                resistance to Round Up herbicide
                                (glyphosate)




   Delta endotoxin crystal
            Bacillus thuringiensis
Clone the
gene
                           Delta endotoxin crystal




                      Bt gene

                                               ori
                       Ti plasmid

                      Ti genes
Engineer   GO                 STOP

the gene




                Bt gene


                                       ori
                 Ti plasmid

                Ti genes
                                     Antibiotic
                                     resistance
Transform           Isolate plant
gene into           cells

plant                                  Grow
                                       undifferentiated
                                       callus




                                    Transform cells




            Select cells

                                                Grow
                                                transgenic
                           Redifferentiate
                                                plant
                           callus
Backcross
GM plant             YYgg x yyGG   YyGg
into high
yield crops
                                   YYgG
                                   YygG
                     YYgg x YyGg
                                   YYgg
GM plant =                         Yygg
yyGG
                                   YYgG
                     YYgG x YYgG
High yield plant =                 YYgg
YYgg                               YYGg
                                   YYGG
  Analysis of
                                                 1   2   3   4   5   6   7


  Results                             GMO
                                      positive
1: non-GMO food with plant primers

2: non-GMO food with GMO primers

3. Test food with plant primers
                                                 1   2   3   4   5   6   7
4: Test food with GMO primers

5: GMO positive template with plant
primers
                                      GMO
6: GMO positive template with GMO
                                      negative
primers
7: PCR MW Ruler
GMO
Investigator Kit
Lab Extensions     • Independent studies

                   • Data Mining/Bioinformatics for
                     specific genes
                      – E.g. Design primers to the cry
                        genes in Bt corn

                   • Quantitative Real-Time PCR
Trouble    • False Positives
shooting     – 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
  Kit Contents                • InstaGene
                              • Master Mix
                              • GMO primers
                              • Plant PSII primers
Not Included but required:    • GMO & PSII positive control DNA
•Thermal cycler
                              • PCR MW Ruler
•Water bath/heat block
•Electrophoresis Module       • DPTPs, microtubes, PCR tubes, foam
(agarose, TAE buffer & Fast     floats
Blast DNA stain)
                              • Manual
•Electrophoresis equipment
& power supply
•2-20 ul pipettes & barrier
tips

				
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posted:4/21/2011
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