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