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Challenges to PCR Biotech Trait Detection

Challenges to PCR Biotech Trait Detection. Satish Rai, Ph.D. Seed Science Center Iowa State University. Seed Science Center. BIGMAP. Seed Pathology. Seed Physiology. Seed International. DNA QA. Seed Conditioning. Computer &Info. Tech. Seed Testing. Curriculum.

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Challenges to PCR Biotech Trait Detection

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

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

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

  4. Approved Events in Corn

  5. 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%

  6. 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)

  7. 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

  8. 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

  9. 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

  10. Sampling Flow Chart Count seed, determine sample size Seed samples Grinding Grind powder

  11. Influence of Particle Size on DNA Extraction Yield Moreano et al. 2005, J. Agric. Food Chem 53:9971-9979

  12. 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

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

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

  15. 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 switchextraction method

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

  17. Issues Related to Quantification of DNA for Quantitative PCR Source: Haque et al., 2003: BMC Biotechnology

  18. Influence of Initial DNA Conc. Qiagen CTAB PMU

  19. 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

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

  21. 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 Cry1A(b) T35S Pepc promo a Cry1A(b) T35S Pepc promo b 35S Bar T35S c

  22. Examples of Some commercial Events Bt 11 has two expression Cassettes Both cassettes contain the 35S promoter Event Bt11 NOS cry1A(b) 35S a NOS Pat 35S b cry1A(b) Mon 810 has only Only copy of promoter and gene sequences 35S Mon 810

  23. 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

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

  25. Examples of Transgenic Events with 35S Promoter Sequences

  26. 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

  27. Challenges in GM Quantification Using 35S Promoter Sequences Event Bt11 NOS cry1A(b) 35S a Bt 11 has two copy of 35S Where as Mon 810 has single Copy of 35S sequences NOS Pat 35S b cry1A(b) 35S Mon 810

  28. 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

  29. 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.

  30. Challenges in GM Quantification with 35S Promoter Sequences

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

  32. 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 Bt gene Bt11 Mon810 Event 176 Design primers from The gene sequences Gene Terminator Promoter

  33. 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

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

  35. Different Copy Number of Gene for Each Event • Event 176 Cry1A(b) Cry1A(b T35S 35S T35S Pepc promo T35S Pepc promo Bar cry1A(b) NK 603 has two Copy of CP4 35S 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

  36. 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

  37. 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 Corn Chromosome Insertion site/Event site

  38. 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

  39. 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.

  40. 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

  41. Thank you

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