1. Regulation of release GMO

2. Safety Assessment The rules governing the assessment of environmental safety of GM crops are still evolving 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 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 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 The environmental safety of directed genetic modification should be evaluated cautiously and thoroughly

3. 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 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 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 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

4. Safety Assessment Appropriate procedures are needed to address any concerns and to identify, assess and minimize the potential risks. 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 National government and international organization have sought to develop regulatory procedures

5. A framework of field testing of GM plants Is the plant a product of classical genetic methods Is the phenotype of the plant equivalent to a product of classical genetic methods? Is the plant modified only by the addition of a DNA sequence that will have no agricultural or environmental effects? Are cross-hybridizing species present? Can the plant escape confinement? Is the genetic modification mobile or otherwise unstable? Performs small scale field test under appropriate containment No Yes Regards as FAMILIAR: Perform field test in accordance with established protocols Perform field test under appropriate containment level based on potential environmental effects Yes or uncertain

6. International aspects of regulation Experiment involving the release of GMO into the environment have been carried out in a number of countries 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 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 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

7. Regulation in the European Community Each release application should contain an analysis and assessment of possible risks 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 The release should only be carried out following the approval of the competent national authority

8. GMO Quantification The EU implements a de minimis threshold for food labelling 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 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

9. Operational Procedures for detection, identification and quantification of GMO’s to comply with labelling regulation of the EU Food Product Detection/ Screening negative positive Identification/ Authorized No YesQuantification Less than 1% No labelling required More than 1% Labelling required Adapted from Anklam et al., 2002 1 2 3 4 5 6

10. Aspects of Quantification Sampling & sample preparation Sampling & sample preparation Procedure determines “representativity” of a resultProcedure determines “representativity” of a result Sample size, homogeneity of the sample and thresholdSample size, homogeneity of the sample and threshold Statistical requirements must be met Statistical requirements must be met Type of material dependent with unique matricesType of material dependent with unique matrices Unprocessed materials eg.grains Unprocessed materials eg.grains Processed ingredients Processed ingredients Processed foods Processed foods

11. Reference Materials Appropriate for positive & negative controls Appropriate for positive & negative controls Basis of validation for analytical procedureBasis of validation for analytical procedure Procedure independentProcedure independent Matrix effects and consistency similar to sample Matrix effects and consistency similar to sample Grains, foods etc…Grains, foods etc… Stable over time, homogeneous & GMO contentStable over time, homogeneous & GMO content DNA methods require numerous +ve controls DNA methods require numerous +ve controls Protein assays only need a single standardProtein assays only need a single standard Institute of Reference Materials and Measurement Institute of Reference Materials and Measurement Geel, BelgiumGeel, Belgium USDA & private companies coming along USDA & private companies coming along Ahmed, 2002

12. Protein Based Testing Methods Transgenes encode for novel proteins Transgenes encode for novel proteins Immunoassay techniques using antibodies Immunoassay techniques using antibodies Ideal for qualitative and quantitative needsIdeal for qualitative and quantitative needs Detects specific proteins in complex matricesDetects specific proteins in complex matrices Analyte must be known Analyte must be known Interference from non-specific interactions of proteins, surfactants (saponins), phenolics, fatty acids and phosphatases Interference from non-specific interactions of proteins, surfactants (saponins), phenolics, fatty acids and phosphatases Polyclonal antibodies Polyclonal antibodies Sensitive but less specific recognitionSensitive but less specific recognition Monoclonal antibodies Monoclonal antibodies Highly specific, slightly less sensitive recognitionHighly specific, slightly less sensitive recognition

13. Western Blot Highly specific qualitative test Highly specific qualitative test Can determine if above or below threshold Can determine if above or below threshold Typically used for research Typically used for research Use denaturing SDS-PAGE Use denaturing SDS-PAGE Solubilizes, removes aggregates & adventitious proteins are eliminatedSolubilizes, removes aggregates & adventitious proteins are eliminated Components of the gel are then transferred to a solid support or transfer membrane Paper towel Transfer membrane Wet filter paper Paper towel weight

14. Western Blot Add monoclonal antibodies Rinse again Antibodies will bind to specified protein Stain the bound antibody for colour development It should look like the gel you started with if a positive reaction occurred Block membrane e.g. dried nonfat milk Block membrane e.g. dried nonfat milk Rinse with ddH 2 O Add antibody against yours with a marker (becomes the antigen)

15. ELISA Microwell Plate Antibody-coated microwells Antibody-coated microwells Quantitative, highly sensitive, economical, high throughput & ideal for laboratory analysisQuantitative, highly sensitive, economical, high throughput & ideal for laboratory analysis Provided non denatured protein Provided non denatured protein Detects 0.25% GMO in seeds & 1.4% toasted mealDetects 0.25% GMO in seeds & 1.4% toasted meal well Antigen Blocking protein Enzyme-labelled Anti- antibody GMO protein specific to antigen Colour Response Concentration Dependent

16. Antibody-coated Tube Similar Approach to ELISA Similar Approach to ELISA Better suited to field testing Better suited to field testing Useful for Qualitative testingUseful for Qualitative testing Quantitative test difficult with no standardsQuantitative test difficult with no standards

17. ELISA variation with immobilized double antibodies, specific to expressed proteins are coupled to a colour reactant and incorporated to a nitrocellulose strip 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 Fast, economical, transferable & good for initial screening Lateral Flow Strip Assay Ahmed, 2002

18. Lateral Flow Strip Protocol Tests for CryI(Ab) & CP4 EPSPS Tests for CryI(Ab) & CP4 EPSPS Plant & seeds Plant & seeds 1 2 3 1.Punch leaf disc 2.Add buffer and grind in tube 3.Insert flow strips for testing

19. New Immunoassay Format Magnetic particles as solid support surface Magnetic particles as solid support surface Coated with capture antibody & placed in tubeCoated with capture antibody & placed in tube Separation using a magnet excludes unbound reactantsSeparation using a magnet excludes unbound reactants Superior kinetics and precision due to uniformitySuperior kinetics and precision due to uniformity VS. Bound Antibodies Free to move in solution

20. ELISA Considerations Large diversity of food matrices need optimization Large diversity of food matrices need optimization Parameters & threshold selection, control tracking and the work environmentParameters & threshold selection, control tracking and the work environment Validation needed Validation needed Extraction efficience, result accuracy, precision, sensitivity (LOD), specificity, reproducibility & consistency/reliabilityExtraction efficience, result accuracy, precision, sensitivity (LOD), specificity, reproducibility & consistency/reliability Standardized Reference Materials!Standardized Reference Materials! Collaborative EU study of 38 laboratories were consistently 0.9% lower than actual amount Collaborative EU study of 38 laboratories were consistently 0.9% lower than actual amount ELISA limited to qualitative detection ELISA limited to qualitative detection

21. DNA-Based Methods Rely on 2 complementary DNA strands that hybridize in a sequence-specific manner Rely on 2 complementary DNA strands that hybridize in a sequence-specific manner rDNA in the crop consists of several unique elements rDNA in the crop consists of several unique elements 35S promoter EPSPS CP4 NOS terminator CaMVAgrobacterium tumefaciens Typically included in rDNA: promoter sequence, structural gene and a stop sequence Diagram representative of a RoundupReady® sequence

22. Sample Preparation - DNA Extraction & Purification of Analytes Extraction & Purification of Analytes Extraction limitations of DNA/proteinExtraction limitations of DNA/protein DNA extraction requirements DNA extraction requirements Lab sample must represent field/food sampleLab sample must represent field/food sample 100-350mg100-350mg Must acquire high quality DNAMust acquire high quality DNA Fragment length and degree of damage Fragment length and degree of damage Heat, low pH, nucleases, depurination, enzymatic degradationHeat, low pH, nucleases, depurination, enzymatic degradation High purity DNAHigh purity DNA Affected by contaminants in food matrices Affected by contaminants in food matrices Polysaccharides, lipids, polyphenols & DNA extraction chemicalsPolysaccharides, lipids, polyphenols & DNA extraction chemicals

23. DNA Extraction Requirements Break cell walls Break cell walls Dry ice or Liquid nitrogenDry ice or Liquid nitrogen Cell membrane disruption Cell membrane disruption Detergents e.g. CTAB or SDSDetergents e.g. CTAB or SDS Inactivate nucleases Inactivate nucleases EDTA binds Mg 2+ & ProteinasesEDTA binds Mg 2+ & Proteinases Separate inhibitory polysaccharides Separate inhibitory polysaccharides Separate Hydrophobic cell constituents Separate Hydrophobic cell constituents E.g.. lipids & polyphenols with organic solventE.g.. lipids & polyphenols with organic solvent Separate from detergent with alcohol Separate from detergent with alcohol

24. Southern Blot Begin by cutting DNA of GMO into fragments with Restriction Enzymes Begin by cutting DNA of GMO into fragments with Restriction Enzymes Run DNA on an agarose gel Run DNA on an agarose gel “Blot” onto a membrane “Blot” onto a membrane Probe membrane Probe membrane Autoradiography Autoradiography +ve-ve P 32 labelled probe

25. Principles of PCR Allows millifold amplification of target DNA Allows millifold amplification of target DNA DNA polymerase makes exact copy of template DNA polymerase makes exact copy of template Synthetic oligonucleotide primers Synthetic oligonucleotide primers Frame desired target sequenceFrame desired target sequence Complimentary to DNAComplimentary to DNA Allows Taq-polymerase enzyme to Allows Taq-polymerase enzyme to generate complimentary sequence generate complimentary sequence between primer sets between primer sets Sequence # grows exponentially Sequence # grows exponentially

26. PCR Fragment Confirmation Gel electrophoresis – size Gel electrophoresis – size Artefact of same size leads to false +ve’sArtefact of same size leads to false +ve’s Southern Blot Assay Southern Blot Assay Reliable but time consumingReliable but time consuming Nested PCR allows for discrimination Nested PCR allows for discrimination 2 nd primer pair within amplicon2 nd primer pair within amplicon Sequencing Sequencing Convenience and time consumingConvenience and time consuming

27. Amplified Fragment Length Polymorphism Originally used to discriminate between and identify various plant varieties Originally used to discriminate between and identify various plant varieties Can identify variety genotypes and low levels of GM DNA Can identify variety genotypes and low levels of GM DNA Detection technique Detection technique possible with GMO- possible with GMO- specific primer and specific primer and identifiable genomic identifiable genomic primer primer

28. Quantitative PCR Testing Normalization of a GM marker to a plant specific reference gene Normalization of a GM marker to a plant specific reference gene Combine 2 absolute quantification reactionsCombine 2 absolute quantification reactions Currently, measure DNA concentration of sample Currently, measure DNA concentration of sample Figure out copy number/genome of both sitesFigure out copy number/genome of both sites = % of GMO’s= % of GMO’s Multiplex PCR Multiplex PCR Both in same reactionBoth in same reaction GM genome-copy/genome-copy ratioGM genome-copy/genome-copy ratio No need for weight or concentration calculationsNo need for weight or concentration calculations

29. Quantitative Competitive PCR Co-amplification of target sequence & internal standard Co-amplification of target sequence & internal standard Corrects for decreased reaction efficiencyCorrects for decreased reaction efficiency Unknown specific target and known control templateUnknown specific target and known control template Optimally want to use same primers that amplify sequences of 2 different sizes Optimally want to use same primers that amplify sequences of 2 different sizes Double QC-PCR uses 2 different reactions Double QC-PCR uses 2 different reactions GM target and trait specificGM target and trait specific E.g. lectin (lel) gene in soybeans, for RRS E.g. lectin (lel) gene in soybeans, for RRS Competitor equivalent to 1% GM soybean (+/-)Competitor equivalent to 1% GM soybean (+/-)

30. QC-PCR Adapted from Hübner et al., 1999

31. Quantitative Real Time PCR PCR amplification exponentially increases until it reaches a plateau between 30 and 40 cycles PCR amplification exponentially increases until it reaches a plateau between 30 and 40 cycles Limiting reaction componentsLimiting reaction components Loss of precision in quantificationLoss of precision in quantification RT-PCR reaction proportional to cycle number RT-PCR reaction proportional to cycle number During exponential phase of PCRDuring exponential phase of PCR Determine known sample point that is same and reference it to the unknown GM fragmentDetermine known sample point that is same and reference it to the unknown GM fragment

33. RT-PCR Allow cycle to cycle monitoring & calculations Allow cycle to cycle monitoring & calculations Quantification by dyes, fluorescent probes, hydrolysis probes & molecular beacons Quantification by dyes, fluorescent probes, hydrolysis probes & molecular beacons Allows for differentiation of artefacts from productAllows for differentiation of artefacts from product A minimum amount of starting sequence DNA is needed – 200ng to detect < 0.1% in maize A minimum amount of starting sequence DNA is needed – 200ng to detect < 0.1% in maize Dependent on genome size and copy numberDependent on genome size and copy number At least 36 copies are desired of GM gene At least 36 copies are desired of GM gene Brodman et al., 2002

34. Summary of detection methods for rDNA products of GM foods Ahmed, 2002