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Challenges to PCR Biotech Trait Detection Satish Rai, Ph.D. Seed Science Center Iowa State University Seed Science Center BIGMAP Seed Pathology DNA QA Seed Physiology Seed International Seed Conditioning Computer &Info. Tech Seed Testing Curriculum Example of Crop with Approved Transgenic Traits Corn Soybean Tomato Potato Rice Cotton Squash Beat Rapeseed/Canola Papaya Flax Tobacco Approved Events in Corn Event Characteristic Bt11 Cry1Ab corn borer resistance + Glufosinate herbicide tolerance Bt176 Cry1Ab corn borer resistance TC1507 Corn borer, black cutworm and armyworm resistance + Glufosinate herbicide tolerance Mon810 Cry1Ab Corn borer protection Mon863 Corn Rootworm Protection MonGA21 Glyphosate herbicide tolerance Nk603 Glyphosate herbicide tolerance T25 Glufosinate herbicide tolerance Mon810+GA21 Cry1Ab corn borer resistance + Glyphosate herbicide tolerance Mon810+Nk603 Cry1Ab Corn borer protection + Glufosinate herbicide tolerance Mon810+T25 Cry1Ab Corn borer protection + Glufosinate herbicide tolerance Mon863+GA21 Corn Rootworm Protection + Glyphosate herbicide tolerance Mon863+NK603 Corn Rootworm Protection + Glyphosate herbicide tolerance Mon 810+Mon 863 (YG Plus) Cry1Ab Corn borer protection + Corn Rootwoom Protection TC1507+NK603 Corn borer, black cutworm and armyworm resistance + Glufosinate herbicide tolerance + Glyphosate Herbicide Tolerance Mon 810+Mon 863+NK603 Cry1Ab Corn borer protection + Corn Rootwoom Protection + Glyphosate Herbicide Tolerance Threshold for Approved GM Traits Japan: 5% Taiwan: 5% Korea: 3% China: Debate is open (0.9% or 3% or 5%) EU: 0.9% US and Canada 5% EU Regulation for GMO Threshold for seeds 0.5% (DNA content) 0.9% in grains 0.5% for unapproved with positive evaluation Screening Event Identification Event Specific Quantification Issues related to current threshold setup by EU (scientific views) Why PCR (DNA) Testing Bioassay and protein test can not be used in some circumstances Testing of breeding samples Testing for approved/unapproved event (backup events) Regulatory requirements Screening for Biotech traits in conventional materials 35S, NOS, NPTII Why Quantitative PCR for seed testing Zygosity Estimate GM content Meet the regulatory compliance in different parts of the world Take advantage of new technology for high throughput applications Challenges in Implementing Quantitative PCR Method for Biotech Trait Quantification Sampling/Grinding DNA extraction method Influence of initial DNA conc Standards Selection of primer/protocol Thresholds Low level detection Higher sensitivity Stacked Trait Hybrid vs. Inbred Ploidy Sampling Flow Chart Seed samples Grinding Count seed, determine sample size Grind powder Influence of Particle Size on DNA Extraction Yield Milling Fraction Particle Sizex50 (uM) CTAB (ng/ul) Wizard Coarse grits Regular grits Meal 1049 196 200 697 173 236 287 320 347 Flour 19 527 359 Moreano et al. 2005, J. Agric. Food Chem 53:9971-9979 Quantification of GM Content from Different Flour Mixes Mix 1: coarse to coarse Mix 2: flour to flour Mix 3: flour to coarse Mix4: coarse to flour Moreano et al. 2005, J. Agric. Food Chem 53:9971-9979 Influence of Sub Sampling on Quantitative Detection Small sub-sample Large Sub sample More particles Better representation Large DNA prep Uniform results Fewer particles More variability Easy DNA extraction Higher Throughput Variability in results Challenges related to DNA Extraction and Impurities at Low Level of GM Detection Charge switch 0.5% CTAB 0.5% Endogenous control Challenges related to DNA Extraction and Impurities at Low Level of GM Detection 1.0, 0.5%, 0.1 R2=0.99 Standard curve prepared using charge switch extraction method Challenges related to DNA Extraction and Impurities at Low Level of GM Detection PMU Extraction Kit: 5%, 2%, 1%, 0.5%, 0.1% Issues Related to Quantification of DNA for Quantitative PCR Evaluatio # DNA n Type Sample conc. (ng/ul OD (UV) 704 20.0 Min (ug/ul) Max (ng/ul) 17.17 24.41 Fl (PG) 5632 5.14 0.00 11.41 Fl (QG) 5632 5.14 0.19 7.95 Influence of Initial DNA Conc. CTAB Qiagen PMU Preparation of Standard for Quantification of Biotech Traits Methods to create standard curve Plasmid DNA with non GM DNA Not recommended DNA/DNA (GM DNA/non GM DNA) This will be very good standard Wt/Wt (create a serial dilution) An alternative to DNA based Seed/Seed (By mixing the seeds) Not suggested Cloned fragments from each events Difficult to find approved standards for all GM traits Strategies for Quantification of Biotech Traits Designing primers from the promoter and terminator sequences Gene specific Event specific Construct specific (used during the transformation) Examples of Some commercial Events Event 176 Pepc promo Cry1A(b) T35S a Cry1A(b) Pepc promo b 35S c Bar T35S T35S Event 176 has three expression Cassettes Two cassettes contain PEPC promoter and two copies of Synthetic cry1A(b) gene Third cassette contains the 35S Promoter sequences Examples of Some commercial Events Event Bt11 a Bt 11 has two expression Cassettes Both cassettes contain the 35S promoter b Mon 810 Mon 810 has only Only copy of promoter and gene sequences Designing Primer Specific to Promoter or Terminator Sequences for GMO Screening Design primer specific to promoter region Need to design several primers to make sure it works with all the events that have promoter region Similarly design primer specific to NOS region Most of commercial agricultural GM products can be detected by using the sequences from 35S promoter and NOS terminator Examples of Transgenic Events with 35S Promoter Sequences Event Name non GM maize Event176 Bt11 T25 Mon810 GA21 NK603 Mon802 MON863 TC1507 Non GM soy GM soy (RR) P35S + + + + + + + + + T-35S + + ? ? - NOS + ? + + + ? ? + Challenges in GM Quantification with 35S Promoter Sequences Very similar to qualitative assay False positive and negative Different version of 35S promoter High degree of homology between CaMV genome and other mosaic viruses common in field crops. Contamination with soil and leaf material Challenges in GM Quantification Using 35S Promoter Sequences Event Bt11 a Bt 11 has two copy of 35S Where as Mon 810 has single Copy of 35S sequences b Mon 810 Challenges in GM Quantification with 35S Promoter Sequences Zygosity level (Homo vs. Hemi) Inbred vs. Hybrids Inbred will always have more GM content than hybrids Challenges in GM Quantification with 35S Promoter Sequences Stacked traits Two or more transgenic traits are stacked together Mon 810+Mon 863+NK603: Cry1Ab Corn borer protection + Corn Rootworm Protection+ Glyphosate Herbicide Tolerance Mon 810 has single copy of 35S NK603 has also single copy of 35S Mon 863 has 2 copy of 35S Total 4 copy of 35S in same hybrid. The expected results with 35 would be somewhere 4 times more than if used with the standard with single copy of 35S. Challenges in GM Quantification with 35S Promoter Sequences 35S copy number 1 copy event 2 copy event Event Trait Bt11 ECB resistance 2 copy 0.2 0.1 Mon810 ECB resistance 1copy 0.1 0.05 TC1507 ECB resistance 1 copy 0.1 0.05 Mon863 CRW 2copy 0.2 0.02 3 copy 0.3 0.15 Mon810+Mon863 ECB +CRW Quantification of Stacked Trait with 35S Mon 810 + NK603 Ct=28 Ct= 31 Mon 810 Ref Ct = 29 Designing Primers and Probe Specific to Gene for Real Time PCR Assay Detection of Transgene Bt gene Bt11 Mon810 Event 176 Design markers for specific gene CP4 gene for roundup Pat and Bar gene for Liberty Design primers from Primer specific to Bt gene The gene sequences Challenges in Designing Primer/Probe for Gene Specific Assay for Quantitative PCR Different forms of the same genes are present Pat and Bar genes Synthetic gene Bt gene Challenges in Designing Primer/Probe for Quantitative PCR Example of cry 1A(b) gene present in three transgenic event Different Copy Number of Gene for Each Event Event 176 Pepc promo Mon 810 T35S Pepc promo T35S 35S Bar T35S NK 603 has two Copy of CP4 Different Copy of number of trait can results in over estimation or underestimation of transgene content when appropriate standard is not available or if event is unknown Challenges in Gene Specific Quantitative PCR Assay Different copy number of the gene can results in over estimations or under estimations of actual GM content Zygosity/ ploidy Primer/Protocol Development for Event Specific Detection Design one primer in the junction region of the insertion site of transgene, and other in the transgene region Design primers flanking to insertion sites GM gene Corn Chromosome Insertion site/Event site Corn Chromosome Challenges in Implementing Event Specific Quantitative PCR Needs to implement multiple step testing strategies to identify events in unknown samples Screening Event identification Quantification Need standards/control for each event If two are more events are stacked, then need to quantify each event separately Why Event Specific Quantification Most of the commercially approved traits/ events are results of single insertion in a given region of genome, thereby resulting in a unique signature site for each transgenes. Results are not influence by copy number of promoter/terminator sequence or copy of transgenes. Regulatory requirements. Ways to Minimize the Variation Sampling, Grinding, Sub sampling Quality of DNA Quantification of Genomic DNA Standards/Control Validation Training New traits with unique DNA sequences Thank you