GMO Investigator Kit Is your food genetically modified

GMO Investigator™ Kit Is your food genetically modified?

GMO Investigator Kit Instructors Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph. D. sherri_andrews@bio-rad. com Essy Levy, M. Sc. essy_levy@bio-rad. com Leigh Brown, M. A. leigh_brown@bio-rad. com

Why teach GMO testing? • Inquiry-based • Real-world test • Environmental Science • Plant Physiology • Genetics and biotechnology • Bioinformatics/Data Mining • Standards-based

GMO Investigator Kit Advantages • Extract and amplify DNA from different food 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 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? Sources: 1996 -1999 Fernandez and Mc. Bride, 2000 -2004: USDA, National Agriculture Statistics Service, Acreage.

Which foods yield viable plant DNA? Very 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

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 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 Ca. MV 35 S and NOS? Ca. MV 35 S – Sequence for the promoter of 35 S 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

Why these steps? Mg++ • Grinding food to release DNA • Insta. Gene chelates divalent ions (e. g. Mg 2+) necessary for DNA degrading enzymes (e. g. DNases) Mg++ Insta. Gene Mg++ • Only 50 μl of food transferred otherwise Insta. Gene is overwhelmed (~ 5 mg of original material) • Boiling releases DNA from food into the Insta. Gene solution • Pellet Insta. Gene and food debris because Insta. Gene 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 èForward çReverse • Nucleotides - d. ATP, d. CTP, d. GTP, d. TTP • Magnesium chloride - enzyme cofactor • Buffer - maintains p. H & contains salt • Taq DNA polymerase – thermophillic enzyme from hot springs

Polymerase Chain Reaction PCR Animation http: //www. bio-rad. com/Life. Science/jobs/2004/04 -0522_PV 92_PCR. html

The PCR Reaction Heat (94 o. C) to denature DNA strands How does it work? Cool (59 o. C) to anneal primers to template Warm (72 o. C) 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 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 • Herbicide. Tolerance: 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 yy. GG Yy. Gg YYgg x Yy. Gg YYg. G Yyg. G YYgg Yygg GM plant = yy. GG High yield plant = YYgg YYg. G x YYg. G 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 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

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 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 – Insta. Gene matrix transferred to PCR reactions

GMO Investigator Kit 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 • Bio-Rad certified Non-GMO food • Insta. Gene • Master Mix • GMO primers • Plant PSII primers • GMO & PSII positive control DNA • PCR MW Ruler • DPTPs, microtubes, PCR tubes, foam floats • Manual

Webinars • Enzyme Kinetics — A Biofuels Case Study • Real-Time PCR — What You Need To Know and Why You Should Teach It! • Proteins — Where DNA Takes on Form and Function • From plants to sequence: a six week college biology lab course • From singleplex to multiplex: making the most out of your realtime experiments explorer. bio-rad. com Support Webinars