PCR Technology for the Detection of Biotechnology Traits: Qualitative, Semi-Quantitative, and Quantitative Methods David Piñero.
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PCR Technology for the Detection of Biotechnology Traits:
Qualitative, Semi-Quantitative, and Quantitative Methods
David Piñero
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
DuPont
Tyvek® flexible sheet products
Supro®isolated soy proteinsCorian®
surfaces
SilverStone®non-stick coatings
CoolMax®performance fibers
Lycra®elastane
Teflon® fabric protector
Mylar®polyester film
• 70th largest US Corporation
• Business in 70 countries
• Employees ~50,000
• Revenues $24 billion
• Net Income $ 2 billion
Kevlar®brand fiber
Stainmaster®carpeting
Sorona®.bio-material
Pioneer Hi-BredPioneer Hi-BredWorld 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
Where are we located?
You can add some pictures,normally they are very helpful to
Give a general idea of the
facilities.
GET Lab Objective: Meeting ALL GMO testing demand…
• Manage and conduct routine PCR testing for Pioneer Supply ManagementTesting for AP in conventional
seed lotsTesting for AP of genetically
modified corn within transgenic corn
Zygosity Testing of transgenic corn
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
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– Refined products exempt
• South Korea:– 3% threshold – Only top 5 ingredients– Refined products exempt
What is Detected
• Common Genetic Construct Elements– 35S promoter– NOS terminator
• Inserted Genes (trait specific)
• Genetic Overlaps (construct specific)
• Insertion Sites (event specific)
DNA Target Sequences for GM Testing:
Generic Transformation Construct:
Gene Fragment
Gene fragment
Inverted Gene
Fragments
Gene Coding
SequencePromoter and intron
Terminator
GeneCoding
Sequence
CAMV 35S
Promoter
CAMV 35S
Terminator
Terminator Fragment
GeneForward Primer
Gene Reverse Primer
Gene Probe
GeneForward
Primer
GeneProbe
CaMV 35S Forward Primer
CaMV 35S Reverse Primer
Gene
Reverse
Primer
Event 35S NOS176 (Maximizer) Yes No
1507 (Herculex) Yes No
B16 Yes No
Bt10 Yes Yes
Bt11 (Agrisure Advantage) Yes Yes
CBH-351 (Starlink) Yes Yes
DAS-06275-8 Yes No
DAS-59122-7 (Herculex RW) Yes No
DBT418 (Bt-Xtra) Yes No
GA21 No Yes
LY038 No No
MIR604 (Agrisure RW) Yes Yes
MON 80100 Yes Yes
MON802 Yes Yes
MON809 Yes Yes
MON810 (Yieldgard) Yes No
MON832 Yes Yes
MON863 Yes Yes
MON88017 Yes Yes
MS3 Yes Yes
MS6 Yes No
NK603 (Roundup Ready) Yes Yes
T14 Yes No
T25 (Liberty Link) Yes No
Steps of the Process
• Sampling• Sub-sampling• DNA Isolation• DNA Quantification • DNA Normalization• PCR• Post-PCR• Data Analysis
DNA Quantification/Normalization
• Fluorometry– Picogreen ®– Hoechst Dye
• UV-Visible Spectrophotometry– AD 260-280
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)
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
SYBR Green Detection
• Dye binds double stranded DNA
• Melting curve analysis
• Qualitative/ Quantitative
• Low specificity
• Generally singleplex
Summary of Probe Formats
SimpleProbe Format:Single fluorescent labeled probe. Fluorescence signal depends on hybridization status (Fluorescein)
HybProbe Format:Dual Probe System utilizing FRET between hybridized labeled probes(Fluos & LightCycler Red 640)
The LightTyper SystemSNP Identification by Melting Curves
Thanks to Louise Gameau, Roche Applied Science
QPCR Data Analysis
4 Types of analysis1. Serial dilution standard curve
(copy number)
2. Serial dilution standard curve (GM %)
3. CT standard curve
4. CT ( CT = CT, sample – CT, calibrator; no standard curve as such)
For 2-4, DNA Normalization is required
PCR efficiency plays an important role
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
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)
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
Validation
• Sensitivity/detectability– LOD– LOQ– Measurement Range
• Accuracy• Precision• Specificity• Robustness & Ruggedness
Convention & Harmonization
• Using the same defined standards
• Using same parameters and converging to acceptable validation practices
• Performance based accreditation.
Proficiency/Check Sample/Accreditation
Programs
• USDA/GIPSA• AOCS• ISTA
Quality Programs
• ISO 9000, 10725• GLP
Harmonization Programs
• ISO TC34 WG7• Codex
Alimentarius• CEN
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