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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 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: – – – – 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 • • • • • • • • 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 • • • • • • • 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 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 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) 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