GMO Investigator Kit

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GMO Investigator Kit

• Instructor
• Sherri Andrews, Ph.D.
• North Carolina School of the Arts
• Winston-Salem, NC

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Why teach GMO testing?

• Inquiry-based
• Real-world test
• Environmental Science
• Plant Physiology
• Genetics and biotechnology
• Bioinformatics/Data Mining

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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

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What is a GMO?

• "genetically modified organism (GMO)" means an
  organism in which the genetic material has been
  altered in a way that does not occur naturally by
  mating and/or natural recombination

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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

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Which foods contain GM product?
Sources 1996-1999 Fernandez and McBride,
2000-2004 USDA, National Agriculture Statistics
Service, Acreage.
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Which foods yield viable plant DNA?
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Why test for GMOs?

• Legislation
• US food labeled GM-Free lt5 GM
• EU food labeled GM if gt1 GM
• Japan food labeled GM if gt5
• Export
• What about unlabeled food?

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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
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How to test for GMOs

• Test for GMOs by PCR
• Grind food
• Extract DNA from sample
• Test sample DNA for viable plant DNA
• Test sample DNA for genetic modifications

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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

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Why amplify a plant gene?
To confirm that viable DNA was extracted and that
negative GM result isnt due to a non-viable
template. Use highly conserved chloroplast gene
from Photosystem II part of the light reaction
of photosynthesis.

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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

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Extract DNA from food
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• Grinding food to release DNA
• InstaGene chelates divalent ions (e.g. Mg2)
  necessary for DNA degrading enzymes (e.g. DNases)
• Only 50 µl of food transferred otherwise
  InstaGene is overwhelmed ( 5 mg of original
  material)
• Boiling releases DNA from food into the InstaGene
  solution
• Pellet InstaGene and food debris because
  InstaGene inhibits PCR reaction (Taq needs Mg)

Why these steps?
Mg
Mg
Mg
Mg
Mg
Mg
Mg
Mg
InstaGene
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Set up PCR reactions
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Volumetric Measurements
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The PCR ReactionWhat do you need?
What is needed for PCR?

• 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

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PCR Review
PCR Animation
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Heat (94oC) to denature DNA strands Cool (59oC)
to anneal primers to template Warm (72oC) to
activate Taq polymerase, which extends primers
and replicates DNA Repeat 40 cycles
The PCR ReactionHow does it work?
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Why have GM crops?

• Growing human population
• Loss of farmable land
• Remediation of soil
• Enrich nutrient content

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Desirable Traits

• Pest Resistance
• Herbicide Tolerance
• Viral Resistance
• Drought Resistance
• Increased Nutritional Value
• Improved Fruit
• Altered Ripening

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Opponents argue

• Creation of super pests
• Creation of super weeds
• Loss of biodiversity
• Biotechnology companies control agriculture
• Health concerns

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Method for Genetic Modification of Crops

• Choose desirable trait
• Clone the gene
• Engineer the gene
• Transform gene into plant
• Backcross GM plant into high yield crops

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Choose desirable trait

• 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)

Bacillus thuringiensis
Delta endotoxin crystal
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Bacillus thuringiensis
Clone the gene
Delta endotoxin crystal
Bt gene
ori
Ti plasmid
Ti genes
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Engineer the gene
Bt gene
ori
Ti plasmid
Ti genes
Antibiotic resistance
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Transform gene into plant
Isolate plant cells
Grow undifferentiated callus
Transform cells
Select cells
Grow transgenic plant
Redifferentiate callus
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Backcross GM plant into high yield crops
YYgg x yyGG
YyGg
YYgG YygG YYgg Yygg
YYgg x YyGg
GM plant yyGG High yield plant YYgg
YYgG YYgg YYGg YYGG
YYgG x YYgG
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1
3
2
7
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Analysis of Results
GMO positive
1 non-GMO food with plant primers
2 non-GMO food with GMO primers
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2
7
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3 Test food with plant primers
4 Test food with GMO primers
GMO negative
5 GMO positive template with plant primers
6 GMO positive template with GMO primers
7 PCR MW Ruler
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GMO Investigator KitLab Extensions

• Independent studies
• Data Mining/Bioinformatics for specific genes
• E.g. Design primers to the cry genes in Bt corn
• Testing for blended foods

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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
• InstaGene matrix transferred to PCR reactions

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GMO Investigator Kit contents

• Bio-Rad certified Non-GMO food
• InstaGene
• Master Mix
• GMO primers
• Plant PSII primers
• GMO PSII positive control DNA
• PCR MW Ruler
• DPTPs, microtubes, PCR tubes, foam floats
• Manual

• 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