1. PCR Technology for the Detection of Biotechnology Traits: Qualitative, Semi-Quantitative, and Quantitative Methods David Piñero

2. Table of Contents Our background Background on GMO testing
What is detected
Introduction on PCR
Types of PCR technologies
Methodology issues
QA/QC
Harmonization
Conclusion/Questions

3. DuPont 70th largest US Corporation Business in 70 countries
CoolMax®
performance fibers
DuPont
Stainmaster®
carpeting
70th largest US Corporation
Business in 70 countries
Employees ~50,000
Revenues $24 billion
Net Income $ 2 billion
Teflon®
fabric protector
Kevlar®
brand fiber
Tyvek®
flexible sheet products
SilverStone®
non-stick coatings
Key Points of slide:
As it begins its third “step change”, DuPont is a large, profitable company known primarily for its petroleum-based fibers, etc.
Consumers know DuPont for these products….
Tie into the Buy DuPont campaign theme?
Mylar®
polyester film
Supro®
isolated soy proteins
Lycra®
elastane
Sorona®.bio-material
Corian®
surfaces

4. Pioneer Hi-Bred World leader in crop genetics
Sales in about 70 countries
~5000 employees
>$2.0 billion in sales 3 yrs in a row
“Delivering Value” is key to future success and ability to meet stakeholder expectations
Over the years, Pioneer has become a very successful company … as the points on the slide illustrate
Pioneer’s success is still directly related to customer success - and to the extent to which they believe we contribute to it.
Pioneer is successful because we have consistently met or exceeded their needs/expectations … and delivered more value to them than our competitors have been able to do.
Our future success depends on our ability to continue this tradition.
To the extent that we can continue to earn ever more of our customers’ business, we will continue to grow …. and meet our stockholders’ employees’ and other stakeholders’ expectations.

5. GET (Genetic Enhancement Testing) Lab, Pioneer Hi-Bred, International

6. Where are we located?
You can add some pictures,normally they are very helpful to
Give a general idea of the facilities.

7. GET Lab Objective: Meeting ALL GMO testing demand…
Manage and conduct routine PCR testing for Pioneer Supply Management
Testing for AP in conventional seed lots
Testing for AP of genetically modified corn within transgenic corn
Zygosity Testing of transgenic corn

8. Why GMO Testing? Labeling Legislations Approved/Un-approved Events
Voluntary Non-GM Labeling
Organic Foods
Testing for Presence of a High-Value Commodity
Genetic Purity Testing
GM within GM
Zygosity Testing

9. Labeling Legislations
European Union:
0.9% threshold
Any ingredient
Imposes traceability
Labeling even if not detectable (i.e. refined oil, corn syrup)
Japan:
5% threshold
Only top 3 ingredients
Refined products exempt
Australia/New Zealand:
1% threshold
South Korea:
3% threshold
Only top 5 ingredients

10. What is Detected Common Genetic Construct Elements
35S promoter
NOS terminator
Inserted Genes (trait specific)
Genetic Overlaps (construct specific)
Insertion Sites (event specific)

11. DNA Target Sequences for GM Testing: Generic Transformation Construct:
Reverse
Primer
Gene
Fragment
Gene fragment
Inverted
Fragments
Coding
Sequence
Promoter
and intron
Terminator
CAMV
35S
Forward
Primer
Reverse
Probe
CaMV 35S

12. Event 35S NOS 176 (Maximizer) Yes No 1507 (Herculex) B16 Bt10
Bt11 (Agrisure Advantage)
CBH-351 (Starlink)
DAS
DAS (Herculex RW)
DBT418 (Bt-Xtra)
GA21
LY038
MIR604 (Agrisure RW)
MON 80100
MON802
MON809
MON810 (Yieldgard)
MON832
MON863
MON88017
MS3
MS6
NK603 (Roundup Ready)
T14
T25 (Liberty Link)

13. Steps of the Process Sampling Sub-sampling DNA Isolation
DNA Quantification
DNA Normalization
PCR
Post-PCR
Data Analysis

14. DNA Quantification/ Normalization
Fluorometry
Picogreen ®
Hoechst Dye
UV-Visible Spectrophotometry AD

15. Automation DNA Isolation DNA Quantification DNA Normalization
PCR Setup

16. PCR 5’ 3’ 5’ 3’
Mg++
Mg++
Mg++

17. Standard PCR Qualitative Lower throughput
Post-PCR step (Agarose Gel Electrophoresis)
Higher contamination risk
Specificity confirmed by size
Bands can be further confirmed (Sequencing/ Restriction Enzymes)

18. Real-Time PCR

19. Real-Time PCR Quantitative Specific (varies with chemistry)
High-Throughput
Eliminates Post-PCR step
Reduces contamination risk
Higher reagent cost
Various chemistries
Sybr Green™
TaqMan™
Scorpion™
Hybridization Probes
Simple Probes
Molecular Beacons
Multiplex capability in some chemistries

20. SYBR Green Detection Dye binds double stranded DNA
Melting curve analysis
Qualitative/ Quantitative
Low specificity
Generally singleplex

21. Top image: Applied Biosystems
Bottom Image: University of North Carolina

22. Molecular Beacons
Copyright: Public Health Research Institute

23. Scorpion
Figure 1. Scorpion probing mechanism. Step 1: initial denaturation of target and Scorpion stem sequence. Step 2: annealing of Scorpion primer to target. Step 3: extension of Scorpion primer produces double-stranded DNA. Step 4: denaturation of double-stranded DNA produced in step 3. This gives a single-stranded target molecule with the Scorpion primer attached. Step 5: on cooling, the Scorpion probe sequence binds to its target in an intramolecular manner. This is favoured over the intermolecular binding of the complementary target strand.

24. Thanks to Louise Gameau, Roche Applied Science
The LightTyper System
SNP Identification by Melting Curves
Summary of Probe Formats
HybProbe Format:
Dual Probe System utilizing FRET between hybridized labeled probes
(Fluos & LightCycler Red 640)
SimpleProbe Format: Single fluorescent labeled probe. Fluorescence signal depends on hybridization status (Fluorescein)
Thanks to Louise Gameau, Roche Applied Science

27. QPCR Data Analysis 4 Types of analysis
Serial dilution standard curve (copy number)
Serial dilution standard curve (GM %)
DCT standard curve
DD CT (DD CT = DCT, sample – DCT, calibrator; no standard curve as such)
For 2-4, DNA Normalization is required
PCR efficiency plays an important role

28. Issues to be Aware of Contamination (genomic DNA or PCR amplicons)
Zygosity
Hybrid status (male/female)
Target copy number
PCR inhibition
Matrix effects
DNA degradation
DNA endoduplication (i.e. seed tissues)
Analytical/instrument error

29. Contamination Control:
Air Control
Aliquoting reagents
Dedicated tools
Radiating plastic ware
Filter tips
Chemical (UNG)
CONTROLS (nulls, NTC, amp. Etc)
Segregation (genomic from PCR, samples from nulls)

30. QA/QC Positive controls Negative controls (NTC)
Nulls (sample prep/DNA Isolation)
Replicates
Amplification controls (spikes and/or endogenous controls)
Acceptance criteria
Standard deviation
Correlation coefficient
PCR efficiency

31. Validation Sensitivity/detectability LOD LOQ Measurement Range
Accuracy
Precision
Specificity
Robustness & Ruggedness

32. Convention & Harmonization
Using the same defined standards
Using same parameters and converging to acceptable validation practices
Performance based accreditation.

33. Proficiency/Check Sample/Accreditation Programs
USDA/GIPSA
AOCS
ISTA
Quality Programs
ISO 9000, 10725
GLP
Harmonization Programs
ISO TC34 WG7
Codex Alimentarius
CEN

34. Thank You