today s pcr gmo detection l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Today’s PCR: GMO Detection PowerPoint Presentation
Download Presentation
Today’s PCR: GMO Detection

Loading in 2 Seconds...

play fullscreen
1 / 35

Today’s PCR: GMO Detection - PowerPoint PPT Presentation


  • 752 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?.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
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
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
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
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 product5
Which foods contain GM product?

Source: USDA ERS http://www.ers.usda.gov/Data/BiotechCrops/

why have gm crops
Why have GM crops?
  • Growing human population
  • Loss of farmable land
  • Remediation of soil
  • Enrich nutrient content
desirable traits
Desirable Traits
  • Pest Resistance
  • Herbicide Tolerance
  • Viral Resistance
  • Drought Resistance
  • Increased Nutritional Value
  • Improved Fruit
  • Altered Ripening
opponents argue
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
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
slide10

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

slide11

Clone the gene

Bacillus thuringiensis

Delta endotoxin crystal

Bt gene

ori

Ti plasmid

Ti genes

slide12

GO

STOP

Engineer the gene

Bt gene

ori

Ti plasmid

Ti genes

Antibiotic resistance

slide13

Transform gene into plant

Isolate plant cells

Grow undifferentiated callus

Transform cells

Select cells

Grow transgenic plant

Redifferentiate callus

slide14

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
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?
slide16

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

slide17

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
slide20

Why these steps?

  • 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
Break to Prepare Food DNA
  • Control food: Mung Bean
  • Unknown foods:
    • ??
    • ??
    • ??
why amplify a plant gene
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
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

slide27

What is needed for PCR?

  • 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
slide28

The PCR Reaction

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
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
slide30

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

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

slide31

Analysis of Results

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

gmo detection pcr set up
GMO Detection: PCR set-up

Today a slight

modification