1. Challenges to PCR Biotech Trait Detection Satish Rai, Ph.D. Seed Science Center Iowa State University

2. Seed Science Center Seed Pathology DNA QA Seed International Seed Physiology Seed Conditioning Computer &Info. Tech Seed Testing Curriculum BIGMAP

7. Example of Crop with Approved Transgenic Traits  Corn  Soybean  Tomato  Potato  Rice  Cotton  Squash  Beat  Rapeseed/Canola  Papaya  Flax  Tobacco

8. 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 Approved Events in Corn

9. 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%

10. 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)

11. 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

12. 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

13. 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

14. Sampling Flow Chart Seed samples Count seed, determine sample size GrindingGrind powder

15. Influence of Particle Size on DNA Extraction Yield Milling Fraction Particle Sizex50 (uM) CTAB (ng/ul) Wizard Coarse grits 1049196200 Regular grits 697173236 Meal287320347 Flour19527359 Moreano et al. 2005, J. Agric. Food Chem 53:9971-9979

16. 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

17. Influence of Sub Sampling on Quantitative Detection Large Sub sample More particles Better representation Large DNA prep Uniform results Small sub-sample Fewer particles More variability Easy DNA extraction Higher Throughput Variability in results

18. Challenges related to DNA Extraction and Impurities at Low Level of GM Detection Charge switch 0.5% CTAB 0.5% Endogenous control

19. Challenges related to DNA Extraction and Impurities at Low Level of GM Detection 1.0, 0.5%, 0.1 R 2 =0.99 Standard curve prepared using charge switch extraction method

20. PMU Extraction Kit: 5%, 2%, 1%, 0.5%, 0.1% Challenges related to DNA Extraction and Impurities at Low Level of GM Detection

21. Issues Related to Quantification of DNA for Quantitative PCR Evaluatio n Type # Sample DNA conc. (ng/ul Min (ug/ul) Max (ng/ul) OD (UV) 70420.017.1724.41 Fl (PG) 56325.140.0011.41 Fl (QG) 56325.140.197.95 Source: Haque et al., 2003: BMC Biotechnology

22. CTAB Qiagen PMU Influence of Initial DNA Conc.

23. 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

24. Strategies for Quantification of Biotech Traits  Designing primers from the promoter and terminator sequences  Gene specific  Event specific  Construct specific (used during the transformation)

25. Examples of Some commercial Events  Event 176 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 Pepc promo Cry1A(b)T35S Pepc promo Cry1A(b) T35S 35SBarT35S a b c

26. Examples of Some commercial Events Event Bt11 Bt 11 has two expression Cassettes Both cassettes contain the 35S promoter Mon 810 Mon 810 has only Only copy of promoter and gene sequences a b

27. 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

28. Most of commercial agricultural GM products can be detected by using the sequences from 35S promoter and NOS terminator

29. Event Name non GM maize Event176 Bt11 T25 Mon810 GA21 NK603 Mon802 MON863 TC1507 Non GM soy GM soy (RR) P35S - + - + - + T-35S - + - + - ? - NOS - + - ? + ? - + Examples of Transgenic Events with 35S Promoter Sequences

30. 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

31. Challenges in GM Quantification Using 35S Promoter Sequences Event Bt11 Mon 810 a b Bt 11 has two copy of 35S Where as Mon 810 has single Copy of 35S sequences

32. 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

33. 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.

34. Challenges in GM Quantification with 35S Promoter Sequences EventTrait 35S copy number 1 copy event 2 copy event Bt11ECB resistance2 copy0.20.1 Mon810ECB resistance1copy0.10.05 TC1507ECB resistance1 copy0.10.05 Mon863CRW2copy0.20.02 Mon810+Mon863ECB +CRW3 copy0.30.15

35. Quantification of Stacked Trait with 35S Ct=28 Ct= 31 Ref Ct = 29 Mon 810 + NK603 Mon 810

36. Designing Primers and Probe Specific to Gene for Real Time PCR Assay  Detection of Transgene  Design markers for specific gene  CP4 gene for roundup  Pat and Bar gene for Liberty  Primer specific to Bt gene Design primers from The gene sequences Bt gene Bt11 Mon810 Event 176

37. 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

38. Challenges in Designing Primer/Probe for Quantitative PCR Source: Matsuoka et al., 2002 Example of cry 1A(b) gene present in three transgenic event

39. Different Copy Number of Gene for Each Event  Event 176 Pepc promoT35SPepc promo T35S35S Bar T35S Mon 810 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 NK 603 has two Copy of CP4

40. 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

41. 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 GM gene Corn Chromosome Insertion site/Event site Design primers flanking to insertion sites

42. 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

43. 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.

44. 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

45. Thank you