OMIC USA OFFERINGS

THRESHOLD FOR APPROVED GM TRAITS

Japan: 5% Taiwan: 5% Korea: 3% China: Open (0.9% or 3% or 5%) EU: 0.9% U.S. & Canada: 5%

PROCESS INVOLVED IN MAKING GMO PLANTS / CRITERIA

Gene discovery Transformation (insertion of novel genes) Good selectable markers Characterization of inserted genes Gene expression analysis Field evaluation and selection Regulatory submission and approval Commercial launch and marketing of new variety

STEPS IN GMO TESTING

SAMPLE LOG IN Inspect, input, and assign a unique number for tracking

SAMPLE PREPARATION Sub-sampling, grinding, homogenization, and DNA

sample collection

STEPS IN GMO TESTING

DNA / PROTEIN EXTRACTION Extract, purify, quantitate, and standardize

DNA AMPLIFICATION BY PCR

Prepare master mix, PCR plate, and run

ANALYSIS OF RESULTS AND REPORTING Review criteria: R², % recovery, slope, efficiency

DNA-GMO DEPARTMENT • Specifically designed and

controlled rooms with positive and negative air pressure environments, thus preventing contamination that might otherwise compromise the lab's sensitive instruments.

SAMPLE PREPARATION • Sub-sampling, weighing, and

grinding • Application of statistical tools • Collection of representative

sample

LABORATORY FACILITIES

OLD TECHNOLOGY NEW TECHNOLOGY

NEW TECHNOLOGY & OPERATIONS

QUALITY CONTROL • Instrument totally clean

between samples • The ZM200 Cyclone minimizes

the emission of dust

DNA EXTRACTION

PROCESS OF DNA EXTRACTION • Incubation of DNA samples and cellular lyses • Pre-purification of DNA • Membrane purification of DNA • Precipitation and elution of DNA

QUALITY CONTROL • Maintain a safe distance between samples

(tubes) • Pipette tips for repetitive use should not

touch the tube • The use of filtered pipette tips

VALIDATION OF DNA • Qubit Fluorometer (DNA-specific dye) • Fluorescence excitation / emission • The method measures dsDNA against • λDNA • Variation (CV) of replicate DNA ≤ 3%

REAL-TIME TAQMAN PCR

REAL-TIME TAQMAN PCR

QUANTITATIVE PCR • Performed in real-time PCR • TaqMan probes • Limit of detection (LOD) 0.01% • Limit of quantification (LOQ) 0.1% • Quantitative: absolute or relative • Standard curves generated using

five levels of matrix (0.05%, 0.1%, 1.0%, 5.0%, and 10.0%)

• Amplification and quantification of traces

• Calculation of % GMO based on the slope and the intercept

QUALITATIVE DNA ANALYSIS WITH AGILENT 2100 BIOANALYZER

QUALITATIVE ASSAY USING PCR & AGILENT 2100 BIOANALYZER • Chips and reagents designed for

sizing and analysis of DNA fragments

• Sizing range: 25-1,000 bp • Sizing accuracy: ± 10% (for ladder

as sample) • Sizing reproducibility: 5% CV (for

ladder as sample) • Quantitation accuracy: 20% CV

(for ladder as sample) • Used in the analysis of PCR and

RT-PCR products

QUALITY CONTROL REPEATABILITY OF A METHOD Testing the precisions under intra-lab conditions Same method Testing repeatability conditions Analysis of calculation of the outcome Application of formula

REPRODUCIBILITY OF A METHOD Testing the precisions under reproducible conditions Same method Different person and different date Comparing the two outcomes

QUALITY CONTROL ACCURACY (TRUENESS AND PRECISION) Close to the true value and high precision

QUALITY CONTROL The process is carried out in duplicate

Extraction PCR

Positive control Extraction PCR

Negative control Extraction PCR

Measurement Uncertainty Corn spike level 0.1% (17.9% RSD; 0.006 bias; 0.046 MU) Corn spike level 0.3% (0.10% RSD; 0.10 bias; 0.076 MU)

APPLICABILITY Scope of the Method should be broad specific

SPECIFICITY Event-specific or marker-specific ACCURACY Within the limit of ±25% interference value PRACTICALITY Availability of equipment, practical hindrances R-SQUARE ≥ 0.96 PCR EFFICIENCY -3.0 ≥ slope ≥ 3.6 RSDR Below 25% over the whole range

LOQ Less than 1/10 of the value of the target concentration

LOD Less than 1/20 of the target concentration TRUENESS Within 25% of the accepted reference value

ACCEPTANCE CRITERIA & PERFORMANCE REQUIREMENTS

CHALLENGES IN PCR DETECTION

TARGET MOLECULES Degraded or absent molecule undetectable Significant effects of processing

CAPTURE MOLECULES MUST BE DEVELOPED Impossible without description of target molecules

CERTIFIED REFERENCE MATERIALS The difficulty to obtain CRMs

GENE STACKING

PARTICIPATION IN PROFICIENCY PROGRAMS

USDA / GIPSA Twice a year covering seven traits and six samples for a total of 42 tests;

corn, soy, rice FAPAS Europe

Twice a year; selective traits Canadian Grain Commission

Twice a year; flax DOW AgroSciences

Once every two years; two corn traits Bayer CropScience

Once every two years; two soybean traits)

Validation is the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled. It is a process and not a result. The purpose of validation is to check whether the method is fit for the purpose. How do we validate the analytical method? By performing an in-house validation. THE VALIDATION PROCESS: Acceptance Criteria (Pre-validation Requirements) Performance Requirements

Development of a new method

Optimization of the method

Completion of the validation process

METHOD VALIDATION PROCESS FOR SPECIFIC GMO EVENTS

VALIDATION OF NEW METHODS GENERAL

Experimental Design Optimize DNA

LIMIT OF DETECTION The Lowest detection Limit Soybean 0.05% in 100ng of total DNA

LIMIT OF QUANTIFICATION Is 0.1% in 100ng of total soybean DNA Based on target DNA over total DNA

MOLECULAR SPECIFICITY Screen the primers /probe for specificity The test of homology

VALIDATION OF NEW METHODS CALIBRATION CURVE / STANDARD CURVE

Five Spike Levels Using Target DNA to Generate a Calibration Curve Produced by plotting the Ct values against the log of target copy no for the

calibration points

DATA ANALYSIS Set the threshold and the baseline crosses the first amplification curve Save the settings

CALCULATION

Use this formula if plasmid DNA is used to generate a standard curve GM % = {target (copy #)/endo copy #*100}

Negative

Double Screen

Certificate of Analysis

35S promoter and/or NOS terminator

positive

Repeat Double Screen with new DNA-Extraction

Quantification of each positive event

Positive Negative

35S + NOS - 35S + NOS + 35S - NOS +

Identification: -GA21 -MIR604

Identification: -Bt11 -NK603 -MON863 -MON88017

Identification: -Event MON810 -Bt 176 -event Herculex -Herculex RW -event T25 -nptII

Maize Analysis Scheme

Thank you.