Transcript PCR Technology - Iowa State University

PCR Technology for the Detection of
Biotechnology Traits: Qualitative,
Semi-Quantitative, and Quantitative
Methods
David Piñero
Table of Contents
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Our background
Background on GMO testing
What is detected
Introduction on PCR
Types of PCR technologies
Methodology issues
QA/QC
Harmonization
Conclusion/Questions
DuPont
CoolMax®
performance fibers
Stainmaster®
carpeting
• 70th largest US Corporation
• Business in 70 countries
Teflon®
fabric protector
Kevlar®
brand fiber
• Employees
~50,000
• Revenues
$24 billion
• Net Income
$ 2 billion
Tyvek®
flexible sheet products
SilverStone®
non-stick coatings
Mylar®
polyester film
Lycra®
elastane
Corian®
surfaces
Sorona®.
bio-material
Supro®
isolated soy proteins
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
GET (Genetic Enhancement Testing) Lab,
Pioneer Hi-Bred, International
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
Management
Testing for AP in conventional
seed lots
Testing for AP of genetically
modified corn within transgenic
corn
Zygosity Testing of transgenic corn
Why GMO Testing?
• Labeling
Legislations
• Approved/Unapproved 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:
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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
Reverse
Gene
Reverse
Primer
Primer
Gene
Probe
Gene
Probe
Gene
Forward
Primer
Gene
Forward
Primer
Gene
Fragment
Inverted
Gene
Fragments
CaMV 35S
Reverse
Primer
CaMV 35S
Forward
Primer
Gene
Promoter
Coding Terminator
and intron Sequence
Gene fragment
Terminator
Fragment
CAMV
Gene
CAMV
35S
Coding
35S
PromoterSequenceTerminator
Event
35S
NOS
176 (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
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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
Automation
• DNA Isolation
• DNA Quantification
• DNA Normalization
• PCR Setup
PCR
5’
3’
3’
5’
Mg++
Mg++
Mg++
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
Real-Time PCR
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Quantitative
Specific (varies with chemistry)
High-Throughput
Eliminates Post-PCR step
Reduces contamination risk
Higher reagent cost
Various chemistries
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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
Top image: Applied
Biosystems
Bottom Image: University of
North Carolina
Molecular Beacons
Copyright: Public Health Research Institute
Scorpion
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
QPCR Data Analysis
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4 Types of analysis
1. Serial dilution standard curve (copy
number)
2. Serial dilution standard curve (GM %)
3. DCT standard curve
4. DD CT (DD CT = DCT, sample – DCT,
calibrator; no standard curve as such)
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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:
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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
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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
Thank You