use of pcr technology for purity testing€¦ · purity testing: three basic needs • genetic...
TRANSCRIPT
Use of PCR Technology for Purity Testing
SCSTDean Hemphill
Quality Testing ManagerDuPont Pioneer
Johnston, Iowa, USA
13/13/2016INSERT SENSITIVITY CAPTION HERE
Introduction & Advice
• Understand the industry you are part of• Embrace new technologies• Keep your career path fluid• Maintain business relationships as companies change• Leadership Advice
• Assemble team of knowledgeable experts• Supply Mindset• Expertise• Aptitude
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Purity Testing: Three Basic Needs
• Genetic Purity• Is the product what we think it is - the correct genetics• Does the product contain “offtype”, “self” or “other”• Genetically fixed
• Trait Purity• Is the trait present • Is the trait homozygous (for inbred)• What level of trait purity
• LLP – Low Level Presence – Non GMO• Adventitious presence of a GMO in a non-GMO product• The presence of an unintended GMO in a GMO
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What Are “Traits”?
• Traits are desirable attributes of a plant• e.g. insect or herbicide resistance; “good” oil composition
of seed; drought tolerance; etc.• Can be natural genes or transgenes
• Transgenes• Desirable genes are artificially introduced into plant
genomes (i.e. not by breeding)• Often derived from other species that can not be bred
with the plant of interest (e.g. MON810 / Bt)• Sometimes human-designed genes (e.g. GAT)
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Commercial Product Testing Descriptions
Quality Analysis Tool
Genetic Purity Isozyme Electrophoresis, SNP, Endpoint PCR, Growout
Trait Purity* ELISA, BioAssay, Endpoint PCR
Trait Confirmation* Endpoint PCR, Chemical Rogue
Zygosity Screen* Endpoint PCR, RT PCR
Low level presence of unintended events*
Lateral flow Strip (ELISA), Endpoint PCR, RT PCR
Trait Detection/Grain* Lateral Flow Strip (ELISA), Endpoint PCR
*For GMO containing products 5
TaqMan Chemistry testing for SNPs
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TaqMan more Detail
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What is ddPCR?
• Droplet Digital PCR is a modification of conventional PCR to allow for the direct quantification of nucleic acids.
• It uses limiting dilution, end-point PCR, and Poisson statistics to yield a direct measure of nucleic acid concentration.
• The number of replicates determines the dynamic range of the assay, precision is not limited by PCR chemistry.
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Limiting Dilution by Droplet
Well contains a mixtureof Template (GMO) and no Template (WT) DNA
PCR reaction is emulsified into individual reactions, limiting the template DNA available in each reaction.
WT
WT
WT
WTWT
WTWT
GMO
GMO
GMO
WTWT
WT
WT
WT
WT
GMO
WT
WT
WT
WT
WT
WTWT
WT
WT
WT
GMO
Using end-point PCR, positive reactions are detected.
WTGMOWT
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Comparison of rtPCR and ddPCRReal-Time PCR Droplet-Digital PCR
Emulsification into ~20,000 rxns
Amplification with Real-Time Machine
Amplification
Analyze with droplet readerYields ~20,000 data points
Analyze sample amplification curve against Standard amplification curves
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Molecular Marker Based Trait and Genetic Purity Testing – SNP
Single Nucleotide Polymorphism (SNP)• A difference in DNA sequence of a single nucleotide
• Replace Isozyme testing for genetic purity
• All parent and commercial corn is transferred to SNP testing worldwide
within the DuPont Pioneer organization
• In humans, 99% of the DNA sequence is identical in all persons; 1% varies• The variation can be a large piece of sequence, or a single nucleotide (SNP)• In human SNPs occur every 100 to 300 bases along the 3-billion-base human
genome
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Single Nucleotide Polymorphisms
Insertion/Deletion G->A SNP
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More Definitions…
• Genome• The sum of all genes of an organism. Each cell contains the entire
genome-worth of DNA.
• Genotype• The sum of all gene versions (“alleles”) of an individual organism
(remember, individuals have some differences in their DNA sequences, such as different SNPs).
• Genotyping• Determining the genotype of an organism by testing the DNA of some of
their cells.
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Chromosomes
DNA and histone proteins are packaged into structures called chromosomes.•Humans: 2 sets of 23 chromosomes (one from the mother, one from the father)•Goldfish: 2 x 50 chromosomes•Soybean: 2 x 20 chromosomes•Maize: 2 x 10 chromosomes
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Genes
•Genes are made up of DNA. •Each chromosome contains many genes.•Each gene has a unique sequence of “letters” (i.e. bases).
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SNP Genotyping
In order to detect a particular SNP in the huge genome,we need to “ amplify” (i.e. make many copies of) that genomic regionbefore probing for the SNP.
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SNP Genotype Scores Interpretation
Individuals with heterozygous A/B alleles
Individuals with homozygous A/A alleles
Individuals with homozygous B/B alleles
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Scoring for Inbred Seed Purity Test
Hybrid control
Inbred control
Inbred control
Contamination
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Example of Pioneer Pedigree Profiles Using 8 SNP Markers
PedigreeInbred A G A T C G G G GInbred B G C A C G G G GInbred C G C A C G G G GInbred D A A T T A T C AInbred E A C T T A T G GInbred F G A A T G T C AInbred G A A T T G T C GInbred H G A A C G G G AInbred I A A A T A T C AInbred J A A T T G T C GInbred K G C T C G G G G
Hybrids12345 A,G A,C A,T C,T A,G G,T C,G A,GInbred K G C T C G G G GInbred I A A A T A T C A
98765 A,G A,C T C,T G G,T C,G GInbred K G C T C G G G GInbred J A A T T G T C G 19
Definition of Inbred Offtypes Based on SNP Scores
• Outcross: • different than expected and at least one marker is heterozygous• if GMO product add traits for confirmation
• Other: • other inbred mix - all marker profiles homozygotes but different
than the expected; • if transgenic traits do not line up
• Segregation: • any individual marker that is not fixed • contain heterozygous individuals
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Inbred Data Analysis -- Outcross
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Inbred Data Analysis -- Other
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Definition of Hybrid Offtypes Based on SNP Scores
• Outcross: • wrong male contribution
• Self: • missing the male or female contribution at all heterozygous
markers
• Other: • unexpected genotype
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Hybrid Data Analysis -- Self and OutcrossJOB G
01
G02
G03
G04
G05
G06
G07
G08
G09
G10
G11
G12
G13
G14
G15 T16
T17
T18
T19
T20
T21
T22
T23
T24
T25
FEMALE G T A A C T C T A C T C C C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
MALE A T T A G C T A G C G C G A T NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
HYBRID G,A T A,T A C,G T,C C,T T,A A,G C T,G C C,G C,A T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G T A A C T C T A C T C C C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G T A A C T C T A C T C C C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A C,T A A C T C,T T A C T C C,G C T NEG NEG POS NEG POS POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NF NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
000002983852 G,A T T,A A C,G T,C C,T A,T G,A C G,T C C,G A,C T NEG NEG NEG NEG NEG POS NEG NEG NEG NEG
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Hybrid Data Analysis –Product Mix, 2 different hybrids
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Why convert?
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Advantages SNP Markers for Seed Testing
• Increased resolution & reliability• Automated & high throughput• Objective scoring and interpretation• Decreased cost per informative datapoint• Trait purity as well as genetic purity can be determined at the
same time on the same seed• Applicable for all crops at all regions• Single inbred profile database used worldwide
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Diagnostic Markers EL vs. SNP -DuPont Pioneer Hybrids Profiled in China
PRODUCTEL Diag Markers
SNP Diag Markers
PRODUCTEL Diag Markers
SNP Diag Markers
X001 0 8 X018 3 7X002 0 12 X019 0 11X003 3 8 X020 0 10X004 0 8 X021 0 9X005 4 11 X022 2 9X006 0 6 X023 2 12X007 0 6 X024 2 10X008 3 9 X025 2 11X009 3 11 X026 1 11X010 0 6 X027 0 6X011 0 7 X028 0 9X012 0 7 X029 2 7X013 0 10 X030 2 7X014 0 9 X031 0 12X015 0 10 X032 4 8X016 3 8 X033 0 9X017 3 6 X034 3 13
•EL diagnostic markers of 0 includes those products with incomplete EL profiles•SNP diagnostic markers out of a standard set of markers across 10 maize chromosomes
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Additional Advantages of SNPs v Isozymes• Workforce
• Training• Availability
• Platform• Scalable (from totally manual to fully automated)• Scalable (test multiple crops on same platform)
• Vendors• Reagent availability
• Startup• MaizeGBD.org
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International Rules for Seed Testing
ISTA rules governing commercial seed lot testing frequencies and requirements
http://www.ingentaconnect.com/content/ista/rules
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Open for Discussion
Thank You
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