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Regulation of release GMO Safety Assessment 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 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 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 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 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 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 Sampling & sample preparation • Procedure determines “representativity” of a result • Sample size, homogeneity of the sample and threshold Statistical requirements must be met • Type of material dependent with unique matrices Unprocessed materials eg.grains Processed ingredients Processed foods Reference Materials Appropriate for positive & negative controls • Basis of validation for analytical procedure • Procedure independent Matrix effects and consistency similar to sample • Grains, foods etc… • Stable over time, homogeneous & GMO content DNA methods require numerous +ve controls • Protein assays only need a single standard Institute of Reference Materials and Measurement • Geel, Belgium USDA & private companies coming along Ahmed, 2002 Protein Based Testing Methods Transgenes encode for novel proteins Immunoassay techniques using antibodies • Ideal for qualitative and quantitative needs • Detects specific proteins in complex matrices 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 Monoclonal antibodies • Highly specific, slightly less sensitive recognition Western Blot Highly specific qualitative test Can determine if above or below threshold Typically used for research Use denaturing SDS-PAGE • 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 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 Antibody-coated microwells • Quantitative, highly sensitive, economical, high throughput & ideal for laboratory analysis 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 Similar Approach to ELISA Better suited to field testing • Useful for Qualitative testing • Quantitative test difficult with no standards Lateral Flow Strip Assay 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 1. Punch leaf disc 2. Add buffer and grind in tube 3. Insert flow strips for testing Tests for CryI(Ab) & CP4 EPSPS Plant & seeds New Immunoassay Format 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 Large diversity of food matrices need optimization • Parameters & threshold selection, control tracking and the work environment Validation needed • Extraction efficience, result accuracy, precision, sensitivity (LOD), specificity, reproducibility & consistency/reliability • Standardized Reference Materials! Collaborative EU study of 38 laboratories were consistently 0.9% lower than actual amount ELISA limited to qualitative detection DNA-Based Methods 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 Extraction & Purification of Analytes • Extraction limitations of DNA/protein 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 Break cell walls • Dry ice or Liquid nitrogen Cell membrane disruption • Detergents e.g. CTAB or SDS Inactivate nucleases • EDTA binds Mg2+ & Proteinases Separate inhibitory polysaccharides Separate Hydrophobic cell constituents • E.g.. lipids & polyphenols with organic solvent Separate from detergent with alcohol Southern Blot 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 Allows millifold amplification of target DNA DNA polymerase makes exact copy of template Synthetic oligonucleotide primers Cycles • Frame desired target sequence • Complimentary to DNA 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 Gel electrophoresis – size • Artefact of same size leads to false +ve’s Southern Blot Assay • Reliable but time consuming Nested PCR allows for discrimination • 2nd primer pair within amplicon Sequencing • Convenience and time consuming Amplified Fragment Length Polymorphism 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 Normalization of a GM marker to a plant specific reference gene • Combine 2 absolute quantification reactions Currently, measure DNA concentration of sample • Figure out copy number/genome of both sites • = % of GMO’s Multiplex PCR • Both in same reaction • GM genome-copy/genome-copy ratio • No need for weight or concentration calculations Quantitative Competitive PCR Co-amplification of target sequence & internal standard • Corrects for decreased reaction efficiency • Unknown specific target and known control template 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 PCR amplification exponentially increases until it reaches a plateau between 30 and 40 cycles • Limiting reaction components • Loss of precision in quantification 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 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