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Challenges to PCR Biotech Challenges to PCR Biotech Trait DetectionTrait Detection
Satish Rai, Ph.D.Satish Rai, Ph.D.
Seed Science CenterSeed Science Center
Iowa State UniversityIowa State University
Seed Science CenterSeed Science Center
Seed Pathology
DNA QA Seed International
Seed Physiology
Seed Conditioning Computer &Info. Tech
Seed Testing Curriculum
BIGMAP
Example of Crop with Approved Example of Crop with Approved Transgenic TraitsTransgenic Traits
CornCorn SoybeanSoybean TomatoTomato PotatoPotato RiceRice CottonCotton SquashSquash BeatBeat Rapeseed/CanolaRapeseed/Canola PapayaPapaya FlaxFlax TobaccoTobacco
Event CharacteristicBt11 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
Threshold for Approved GM TraitsThreshold for Approved GM Traits
Japan: 5%Japan: 5% Taiwan: 5%Taiwan: 5% Korea: 3%Korea: 3% China: Debate is open (0.9% or 3% or 5%)China: Debate is open (0.9% or 3% or 5%) EU: 0.9%EU: 0.9% US and Canada 5%US and Canada 5%
EU Regulation for GMO EU Regulation for GMO
Threshold for seeds 0.5% (DNA content)Threshold for seeds 0.5% (DNA content) 0.9% in grains0.9% in grains 0.5% for unapproved with positive 0.5% for unapproved with positive
evaluationevaluation ScreeningScreening Event IdentificationEvent Identification Event Specific QuantificationEvent Specific Quantification
Issues related to current threshold setup Issues related to current threshold setup by EU (scientific views)by EU (scientific views)
Why PCR (DNA) TestingWhy PCR (DNA) Testing
Bioassay and protein test can not be used Bioassay and protein test can not be used in some circumstancesin some circumstances
Testing of breeding samplesTesting of breeding samples Testing for approved/unapproved event Testing for approved/unapproved event
(backup events)(backup events) Regulatory requirementsRegulatory requirements Screening for Biotech traits in conventional Screening for Biotech traits in conventional
materialsmaterials 35S, NOS, NPTII 35S, NOS, NPTII
Why Quantitative PCR for seed Why Quantitative PCR for seed testingtesting
Zygosity Zygosity Estimate GM contentEstimate GM content Meet the regulatory compliance in different Meet the regulatory compliance in different
parts of the worldparts of the world Take advantage of new technology for Take advantage of new technology for
high throughput applicationshigh throughput applications
Challenges in Implementing Quantitative Challenges in Implementing Quantitative PCR Method for Biotech Trait QuantificationPCR Method for Biotech Trait Quantification
Sampling/Grinding Sampling/Grinding DNA extraction methodDNA extraction method Influence of initial DNA concInfluence of initial DNA conc StandardsStandards Selection of primer/protocolSelection of primer/protocol ThresholdsThresholds
Low level detectionLow level detection Higher sensitivityHigher sensitivity
Stacked TraitStacked Trait Hybrid vs. InbredHybrid vs. Inbred PloidyPloidy
Sampling Flow Chart
Seed samples
Count seed, determine sample size
Grinding Grind powder
Influence of Particle Size on DNA Extraction Influence of Particle Size on DNA Extraction Yield Yield
Milling Milling FractionFraction
Particle Particle Sizex50 Sizex50 (uM)(uM)
CTAB (ng/ul)CTAB (ng/ul) WizardWizard
Coarse Coarse gritsgrits
10491049 196196 200200
Regular Regular gritsgrits
697697 173173 236236
MealMeal 287287 320320 347347
FlourFlour 1919 527527 359359
Moreano et al. 2005, J. Agric. Food Chem 53:9971-9979
Quantification of GM Content from Quantification of GM Content from Different Flour Mixes Different Flour Mixes
Mix 1: coarse to coarseMix 2: flour to flourMix 3: flour to coarseMix4: coarse to flour
Moreano et al. 2005, J. Agric. Food Chem 53:9971-9979
Influence of Sub Sampling on Influence of Sub Sampling on Quantitative DetectionQuantitative Detection
Large Sub sampleMore particles
Better representationLarge DNA prepUniform results
Small sub-sampleFewer particlesMore variabilityEasy DNA extractionHigher ThroughputVariability in results
Challenges related to DNA Extraction and Challenges related to DNA Extraction and Impurities at Low Level of GM DetectionImpurities at Low Level of GM Detection
Charge switch 0.5% CTAB
0.5%
Endogenous control
Challenges related to DNA Extraction and Challenges related to DNA Extraction and Impurities at Low Level of GM DetectionImpurities at Low Level of GM Detection
1.0, 0.5%, 0.1
R2=0.99
Standard curve prepared using charge switch extraction method
PMU Extraction Kit: 5%, 2%, 1%, 0.5%, 0.1%
Challenges related to DNA Extraction and Impurities Challenges related to DNA Extraction and Impurities at Low Level of GM Detectionat Low Level of GM Detection
Issues Related to Quantification of Issues Related to Quantification of DNA for Quantitative PCR DNA for Quantitative PCR
EvaluatioEvaluation Typen Type
# # SampleSample
DNA DNA conc. conc. (ng/ul(ng/ul
Min Min (ug/ul)(ug/ul)
Max Max (ng/ul)(ng/ul)
OD (UV)OD (UV) 704704 20.020.0 17.1717.17 24.4124.41
Fl (PG)Fl (PG) 56325632 5.145.14 0.000.00 11.4111.41
Fl (QG)Fl (QG) 56325632 5.145.14 0.190.19 7.957.95
Source: Haque et al., 2003: BMC Biotechnology
CTABQiagen
PMU
Influence of Initial DNA Conc.Influence of Initial DNA Conc.
Preparation of Standard for Quantification of Preparation of Standard for Quantification of
Biotech TraitsBiotech Traits Methods to create standard curveMethods to create standard curve
Plasmid DNA with non GM DNAPlasmid DNA with non GM DNA Not recommended Not recommended
DNA/DNA (GM DNA/non GM DNA)DNA/DNA (GM DNA/non GM DNA) This will be very good standardThis will be very good standard
Wt/Wt (create a serial dilution)Wt/Wt (create a serial dilution) An alternative to DNA basedAn alternative to DNA based
Seed/Seed (By mixing the seeds)Seed/Seed (By mixing the seeds) Not suggestedNot suggested
Cloned fragments from each eventsCloned fragments from each events
Difficult to find approved standards for all GM traitsDifficult to find approved standards for all GM traits
Strategies for Quantification of Strategies for Quantification of Biotech TraitsBiotech Traits
Designing primers from the promoter and Designing primers from the promoter and terminator sequencesterminator sequences
Gene specificGene specific Event specificEvent specific Construct specific (used during the Construct specific (used during the
transformation)transformation)
Examples of Some commercial Examples of Some commercial EventsEvents
Event 176Event 176 Event 176 has three expression CassettesTwo cassettes contain PEPC promoter and two copies of Synthetic cry1A(b) gene
Third cassette contains the 35SPromoter sequences
Pepc promo Cry1A(b) T35S
Pepc promo Cry1A(b) T35S
35S Bar T35S
a
b
c
Examples of Some commercial Examples of Some commercial EventsEvents
Event Bt11 Bt 11 has two expression CassettesBoth cassettes contain the 35S promoter
Mon 810Mon 810 has only Only copy of promoter and gene sequences
a
b
Designing Primer Specific to Promoter or Designing Primer Specific to Promoter or Terminator Sequences for GMO Terminator Sequences for GMO
ScreeningScreening
Design primer specific to promoter region Design primer specific to promoter region Need to design several primers to make sure Need to design several primers to make sure
it works with all the events that have promoter it works with all the events that have promoter regionregion
Similarly design primer specific to NOS Similarly design primer specific to NOS regionregion
Most of commercial agricultural GM products can be detected by using the sequences from 35S promoter and NOS terminator
Event Namenon GM maize
Event176Bt11T25
Mon810GA21NK603
Mon802MON863TC1507
Non GM soyGM soy (RR)
P35S-++++-++++-+
T-35S-+ -+-- - -??--
NOS--+-?+++??-+
Examples of Transgenic Events Examples of Transgenic Events with 35S Promoter Sequenceswith 35S Promoter Sequences
Challenges in GM Quantification Challenges in GM Quantification with 35S Promoter Sequenceswith 35S Promoter Sequences
Very similar to qualitative assayVery similar to qualitative assay False positive and negativeFalse positive and negative
Different version of 35S promoterDifferent version of 35S promoter
High degree of homology between CaMV High degree of homology between CaMV genome and other mosaic viruses common in genome and other mosaic viruses common in field crops.field crops.
Contamination with soil and leaf materialContamination with soil and leaf material
Challenges in GM Quantification Challenges in GM Quantification Using 35S Promoter SequencesUsing 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
Challenges in GM Quantification Challenges in GM Quantification with 35S Promoter Sequenceswith 35S Promoter Sequences
Zygosity level (Homo vs. Hemi)Zygosity level (Homo vs. Hemi) Inbred vs. HybridsInbred vs. Hybrids
Inbred will always have more GM content than Inbred will always have more GM content than hybridshybrids
Challenges in GM Quantification Challenges in GM Quantification with 35S Promoter Sequenceswith 35S Promoter Sequences
Stacked traitsStacked traits Two or more transgenic traits are stacked Two or more transgenic traits are stacked
togethertogether 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 Challenges in GM Quantification with 35S Promoter Sequenceswith 35S Promoter Sequences
Event Trait35S copy
number1 copy
event2 copy
event
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
Mon810+Mon863 ECB +CRW 3 copy 0.3 0.15
Quantification of Stacked Trait with Quantification of Stacked Trait with 35S 35S
Ct=28Ct= 31
Ref Ct = 29
Mon 810 + NK603
Mon 810
Designing Primers and Probe Specific to Designing Primers and Probe Specific to Gene for Real Time PCR AssayGene for Real Time PCR Assay
Detection of TransgeneDetection of Transgene Design markers for specific geneDesign markers for specific gene
CP4 gene for roundupCP4 gene for roundup Pat and Bar gene for LibertyPat and Bar gene for Liberty Primer specific to Bt gene Primer specific to Bt gene
Design primers from The gene sequences
Bt geneBt11Mon810Event 176
Challenges in Designing Primer/Probe for Challenges in Designing Primer/Probe for
Gene Specific Assay for Quantitative PCRGene Specific Assay for Quantitative PCR
Different forms of the same genes are Different forms of the same genes are present present Pat and Bar genes Pat and Bar genes
Synthetic geneSynthetic gene Bt gene Bt gene
Challenges in Designing Challenges in Designing Primer/Probe for Quantitative PCRPrimer/Probe for Quantitative PCR
Source: Matsuoka et al., 2002
Example of cry 1A(b) gene present in three transgenic event
Different Copy Number of Gene for Different Copy Number of Gene for Each EventEach Event
Event 176Event 176
Pepc promo T35S Pepc promo T35S 35SBar
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
Challenges in Gene Specific Challenges in Gene Specific Quantitative PCR AssayQuantitative PCR Assay
Different copy number of the gene can Different copy number of the gene can results in over estimations or under results in over estimations or under estimations of actual GM contentestimations of actual GM content
Zygosity/ ploidyZygosity/ ploidy
Primer/Protocol Development for Primer/Protocol Development for Event Specific DetectionEvent Specific Detection
Design one primer in the junction region of the Design one primer in the junction region of the insertion site of transgene, and other in the insertion site of transgene, and other in the transgene regiontransgene region
GM gene
Corn ChromosomeCorn Chromosome
Insertion site/Event site
Design primers flanking to insertion sites
Challenges in Implementing Event Challenges in Implementing Event Specific Quantitative PCRSpecific Quantitative PCR
Needs to implement multiple step testing Needs to implement multiple step testing strategies to identify events in unknown strategies to identify events in unknown samplessamples Screening Event identification Screening Event identification
QuantificationQuantification
Need standards/control for each eventNeed standards/control for each event If two are more events are stacked, then If two are more events are stacked, then
need to quantify each event separatelyneed to quantify each event separately
Why Event Specific QuantificationWhy Event Specific Quantification
Most of the commercially approved traits/ Most of the commercially approved traits/ events are results of single insertion in a events are results of single insertion in a given region of genome, thereby resulting given region of genome, thereby resulting in a unique signature site for each in a unique signature site for each transgenes. transgenes.
Results are not influence by copy number Results are not influence by copy number of promoter/terminator sequence or copy of of promoter/terminator sequence or copy of transgenes. transgenes.
Regulatory requirements.Regulatory requirements.
Ways to Minimize the VariationWays to Minimize the Variation
Sampling, Grinding, Sub samplingSampling, Grinding, Sub sampling Quality of DNA Quality of DNA Quantification of Genomic DNAQuantification of Genomic DNA Standards/ControlStandards/Control ValidationValidation TrainingTraining New traits with unique DNA sequencesNew traits with unique DNA sequences
Thank youThank you