Transcript RT-PCR - Faperta UGM

Regulation of release GMO
Safety Assessment
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The rules governing the assessment of
environmental safety of GM crops are still
evolving
Product regulation should be based on the
phenotype of the plant rather than on its method
of construction
National and international authorities have
generally taken the view that the release into the
environment of products of certain technique
should be subject to specific regulation
The environmental safety of directed genetic
modification should be evaluated cautiously and
thoroughly
Safety Assessment
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Concerns revolve primarily around the ability of GMO to
replicate, from which it can be argued that any adverse
effects would be multiplied and become uncontrollable
For this reason, the authorities have adopted a cautious,
case by case, and step by step approach
The specific concerns in terms of effect on the environment
can be summarized as 3 questions
1. Will the process of genetic modification make the plant more
persistent in the environment or more invasive of natural habitats
2. Will the GM plant pass on the inserted gene by pollen transfer or
any other means in such a way that other plants become more
persistent or invasive
3. Will the GM plant, or any plants to which the relevant genes
have been transferred, have an additional character which could
lead to detrimental effects in the environment
Safety Assessment
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Appropriate procedures are needed to address any concerns
and to identify, assess and minimize the potential risks.
National government and international organization have
sought to develop regulatory procedures
A framework of field testing of GM
plants
Is the plant a product of classical
genetic methods
Yes
No
Is the phenotype of the plant
equivalent to a product of
classical genetic methods?
Regards as FAMILIAR:
Yes
No
Is the plant modified only by the
addition of a DNA sequence that
will have no agricultural or
environmental effects?
No
Are cross-hybridizing species
present?
No
Can the plant escape
confinement?
Yes
Yes or
uncertain
Yes or
uncertain
No
Is the genetic modification mobile
or otherwise unstable?
No
Performs small scale field
test under appropriate
containment
Perform field test in
accordance with
established protocols
Yes or
uncertain
Perform field test under
appropriate containment
level based on potential
environmental effects
International aspects of
regulation
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Experiment involving the release of GMO into the
environment have been carried out in a number of
countries
Each countries has developed its own procedures and
policies in regulating GMO field trials, usually with
existing legislation as the basis
GMO applications generally should be assessed on a
case by case basis by an expert group of scientists and
that the development of GMO product should be carried
out in a stepwise manner moving from the laboratory
to the growth chamber, to the greenhouse, to limited
field testing, to large scale field testing
Regulation in the European
Community
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Each release application should contain an analysis and
assessment of possible risks
The release should only be carried out following the
approval of the competent national authority
GMO Quantification
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The EU implements a de minimis threshold for food
labelling
The “threshold regulation” specifies that food
stuffs must be subject to labelling where material
derived from GMO’s is present in food ingredients I
a proportion above 1% of the food ingredients
individually considered
Operational Procedures for detection,
identification and quantification of GMO’s
to comply with labelling regulation of the
EU
2
1
Detection/
Food Product
Screening
negative 3
positive
Identification/
4
Authorized
More than 1%
Labelling required
5
Quantification
6
Yes
No
Less than 1%
No labelling required
Adapted from Anklam et al., 2002
Aspects of Quantification
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Sampling & sample preparation
• Procedure determines “representativity” of a result
• Sample size, homogeneity of the sample and threshold
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Statistical requirements must be met
• Type of material dependent with unique matrices
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Unprocessed materials eg.grains
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Processed ingredients
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Processed foods
Reference Materials
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Appropriate for positive & negative controls
• Basis of validation for analytical procedure
• Procedure independent
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Matrix effects and consistency similar to sample
• Grains, foods etc…
• Stable over time, homogeneous & GMO content
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DNA methods require numerous +ve controls
• Protein assays only need a single standard
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Institute of Reference Materials and Measurement
• Geel, Belgium
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USDA & private companies coming along
Ahmed, 2002
Protein Based Testing Methods
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Transgenes encode for novel proteins
Immunoassay techniques using antibodies
• Ideal for qualitative and quantitative needs
• Detects specific proteins in complex matrices
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Analyte must be known
Interference from non-specific interactions of proteins,
surfactants (saponins), phenolics, fatty acids and phosphatases
Polyclonal antibodies
• Sensitive but less specific recognition
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Monoclonal antibodies
• Highly specific, slightly less sensitive recognition
Western Blot
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Highly specific qualitative test
Can determine if above or below threshold
Typically used for research
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Use denaturing SDS-PAGE
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• Solubilizes, removes aggregates & adventitious proteins
are eliminated
Components of the gel are then transferred to a
solid support or transfer membrane
weight
Transfer
membrane
Paper towel
Paper towel
Wet filter paper
Western Blot
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Block membrane e.g. dried nonfat milk
Rinse with ddH2O
Add monoclonal
antibodies
Rinse again
Antibodies will bind to specified protein
Add antibody against yours with a marker (becomes the antigen)
Stain the bound antibody for colour development
It should look like the gel you started
with if a positive reaction occurred
ELISA Microwell Plate
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Antibody-coated microwells
• Quantitative, highly sensitive, economical, high
throughput & ideal for laboratory analysis
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Provided non denatured protein
• Detects 0.25% GMO in seeds & 1.4% toasted meal
Enzyme-labelled Antiantibody
Colour
Response
well Concentration
Dependent
GMO protein
specific to antigen
Blocking
protein
Antigen
Antibody-coated Tube
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Similar Approach to ELISA
Better suited to field testing
• Useful for Qualitative testing
• Quantitative test difficult with no standards
Lateral Flow Strip Assay
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ELISA variation with immobilized double antibodies, specific
to expressed proteins are coupled to a colour reactant and
incorporated to a nitrocellulose strip
Fast, economical, transferable & good for initial screening
Ahmed, 2002
Lateral Flow Strip Protocol
1
2
3
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1. Punch leaf disc
2. Add buffer and
grind in tube
3. Insert flow strips
for testing
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Tests for
CryI(Ab) & CP4
EPSPS
Plant & seeds
New Immunoassay Format
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Magnetic particles as solid support surface
• Coated with capture antibody & placed in tube
• Separation using a magnet excludes unbound
reactants
• Superior kinetics and precision due to uniformity
VS.
Bound Antibodies
Free to move in
solution
ELISA Considerations
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Large diversity of food matrices need optimization
• Parameters & threshold selection, control tracking and
the work environment
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Validation needed
• Extraction efficience, result accuracy, precision,
sensitivity (LOD), specificity, reproducibility &
consistency/reliability
• Standardized Reference Materials!
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Collaborative EU study of 38 laboratories were
consistently 0.9% lower than actual amount
ELISA limited to qualitative detection
DNA-Based Methods
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Rely on 2 complementary DNA
strands that hybridize in a
sequence-specific manner
rDNA in the crop consists of
several unique elements
35S promoter
CaMV
EPSPS CP4
NOS terminator
Agrobacterium tumefaciens
Typically included in rDNA: promoter sequence, structural gene and
a stop sequence
Diagram representative of a
RoundupReady® sequence
Sample Preparation - DNA
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Extraction & Purification of Analytes
• Extraction limitations of DNA/protein
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DNA extraction requirements
• Lab sample must represent field/food sample
• 100-350mg
• Must acquire high quality DNA
 Fragment length and degree of damage
• Heat, low pH, nucleases, depurination, enzymatic
degradation
• High purity DNA
 Affected by contaminants in food matrices
• Polysaccharides, lipids, polyphenols & DNA extraction
chemicals
DNA Extraction Requirements
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Break cell walls
• Dry ice or Liquid nitrogen
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Cell membrane disruption
• Detergents e.g. CTAB or SDS
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Inactivate nucleases
• EDTA binds Mg2+ & Proteinases
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Separate inhibitory polysaccharides
Separate Hydrophobic cell constituents
• E.g.. lipids & polyphenols with organic solvent
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Separate from detergent with alcohol
Southern Blot
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Begin by cutting DNA of GMO into
fragments with Restriction Enzymes
Run DNA on an agarose gel
“Blot” onto a membrane
Probe membrane
Autoradiography
P32 labelled
probe
+ve
-ve
Principles of PCR
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Allows millifold amplification of target DNA
DNA polymerase makes exact copy of template
Synthetic oligonucleotide primers
Cycles
• Frame desired target sequence
• Complimentary to DNA
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Allows Taq-polymerase enzyme to
generate complimentary sequence
between primer sets
Sequence # grows exponentially
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
DNA molecules formed
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
PCR Fragment Confirmation
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Gel electrophoresis – size
• Artefact of same size leads to false +ve’s
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Southern Blot Assay
• Reliable but time consuming
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Nested PCR allows for discrimination
• 2nd primer pair within amplicon
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Sequencing
• Convenience and time consuming
Amplified Fragment Length
Polymorphism
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Originally used to discriminate between and
identify various plant varieties
Can identify variety genotypes and low levels of GM
DNA
Detection technique
possible with GMOspecific primer and
identifiable genomic
primer
Quantitative PCR Testing
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Normalization of a GM marker to a plant specific
reference gene
• Combine 2 absolute quantification reactions
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Currently, measure DNA concentration of sample
• Figure out copy number/genome of both sites
• = % of GMO’s
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Multiplex PCR
• Both in same reaction
• GM genome-copy/genome-copy ratio
• No need for weight or concentration calculations
Quantitative Competitive PCR
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Co-amplification of target sequence & internal
standard
• Corrects for decreased reaction efficiency
• Unknown specific target and known control template
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Optimally want to use same primers that amplify
sequences of 2 different sizes
Double QC-PCR uses 2 different reactions
• GM target and trait specific
 E.g. lectin (lel) gene in soybeans, for RRS
• Competitor equivalent to 1% GM soybean (+/-)
QC-PCR
Adapted from Hübner et al., 1999
Quantitative Real Time PCR
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PCR amplification exponentially increases until it
reaches a plateau between 30 and 40 cycles
• Limiting reaction components
• Loss of precision in quantification
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RT-PCR reaction proportional to cycle number
• During exponential phase of PCR
• Determine known sample point that is same and
reference it to the unknown GM fragment
RT-PCR
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Allow cycle to cycle monitoring & calculations
Quantification by dyes, fluorescent probes,
hydrolysis probes & molecular beacons
• Allows for differentiation of artefacts from
product
A minimum amount of starting sequence DNA is
needed – 200ng to detect < 0.1% in maize
• Dependent on genome size and copy number
 At least 36 copies are desired of GM gene
Brodman et al., 2002
Summary of detection methods for
rDNA products of GM foods
Parameter
Western blot
ELISA
Difficult
moderate
Simple
Difficult
Difficult
Difficult
Difficult
Yes
Yes
No
Yes
Yes
Yes
Yes
Sensitivity
High
High
High
Moderate
Very High
High
High
Duration
2d
30 - 90 min
10 min
6h
1.5d
2d
1d
Cost/sample
150
5
2
150
250
350
450
Quantitative
No
Yes
No
No
No
Yes
Yes
Field application
No
Yes
Yes
No
No
No
No
Field Testing
Academic lab
Test facility
Ease of use
Special equipment
needed
Where applied
Academic lab Test facility
Lateral flow strip Southern blot Qualitative PCR QC-PCR
RT-PCR
Test facility Test facility
Ahmed, 2002