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

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

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 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

Automation

• DNA Isolation

• DNA Quantification

• DNA Normalization

• PCR Setup

PCR

5’ 3’

5’3’

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• 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

Top image: Applied Biosystems

Bottom Image: University of North Carolina

Molecular Beacons

Copyright: Public Health Research Institute

Scorpion

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

Thank You