OMIC USA OFFERINGS 5% 5% 3% Open (0.9% or 3% or 5%) 0.9% 5% - - PowerPoint PPT Presentation

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
• f 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.