Download
Skip this Video
1 / 35
Today’s PCR: GMO Detection - PowerPoint PPT Presentation
- 768 Views
- Uploaded on
Today’s PCR: GMO Detection. What are GM crops? Detecting GM crops Elisa, PCR PCR Analysis Sequences detected (PSII, 35S, NOS) Controls (non-GM, GM-positive) Reaction sets. GMO Investigator Kit. What is a GMO?.
Download Presentation 
An Image/Link below is provided (as is) to download presentation
PowerPoint Slideshow about 'Today’s PCR: GMO Detection' - pauline
An Image/Link below is provided (as is) to download presentation
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.
While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Today’s PCR: GMO Detection
- What are GM crops?
- Detecting GM crops
- Elisa, PCR
- PCR Analysis
- Sequences detected (PSII, 35S, NOS)
- Controls (non-GM, GM-positive)
- Reaction sets
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
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?
Source: USDA ERS http://www.ers.usda.gov/Data/BiotechCrops/
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
- Loss of biodiversity
- Creation of super weeds
- Creation of super pests
- Biotechnology companies control agriculture
- Health concerns
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
- 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
Isolate plant cells
Grow undifferentiated callus
Transform cells
Select cells
Grow transgenic plant
Redifferentiate callus
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
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?
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
- Test for GMOs by PCR:
- Grind food
- Extract DNA from sample
- Test sample DNA for viable plant DNA
- Test sample DNA for genetic modifications
- 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++)
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
Mg++
InstaGene
Break to Prepare Food DNA
- Control food: Mung Bean
- Unknown foods:
- ??
- ??
- ??
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.
How is the GMO DNA detected?
CaMV 35S – Sequence for the promoter of 35S transcript of the Cauliflower mosaic virus.
Used in transgenics because it functions in every plant cell
NOS- Sequence fornopaline synthase terminator from soil bacterium Agrobacterium tumefacians
Used in transgenics because it evolved to be recognized in most plants
- Template - the DNA to be amplified
- Primers - 2 short specific pieces of DNA whose sequence flanksthe 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
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
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
1
2
3
4
5
6
7
GMO positive
1: non-GMO food with plant primers
2: non-GMO food with GMO primers
1
2
3
4
5
6
7
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
1: non-GMO food with plant primers
1
2
3
4
5
6
7
2: non-GMO food with GMO primers
3: Test food with plant primers
4: Test food with GMO primers
5: GMO positive template with plant primers
6: GMO positive template with GMO primers
7: PCR MW Ruler
10ul of Orange Dye into each PCR tube
Load 20ul of PCR Reaction + Dye to each lane
Marker has loading dye – load 20ul
