Transcript Real Time PCR Testing for Biotech Crops: Issues

Challenges to PCR Biotech
Trait Detection
Satish Rai, Ph.D.
Seed Science Center
Iowa State University
Seed Science Center
BIGMAP
Seed Pathology
DNA QA
Seed Physiology
Seed International
Seed Conditioning
Computer &Info. Tech
Seed Testing
Curriculum
Example of Crop with Approved
Transgenic Traits
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Corn
Soybean
Tomato
Potato
Rice
Cotton
Squash
Beat
Rapeseed/Canola
Papaya
Flax
Tobacco
Approved Events in Corn
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
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%
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)
Why PCR (DNA) Testing
 Bioassay and protein test can not be used
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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
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
Challenges in Implementing Quantitative
PCR Method for Biotech Trait Quantification
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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
Sampling Flow Chart
Seed samples
Grinding
Count seed, determine
sample size
Grind powder
Influence of Particle Size on DNA Extraction
Yield
Milling
Fraction
Particle
Sizex50
(uM)
CTAB (ng/ul) Wizard
Coarse
grits
Regular
grits
Meal
1049
196
200
697
173
236
287
320
347
Flour
19
527
359
Moreano et al. 2005, J. Agric. Food Chem 53:9971-9979
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
Influence of Sub Sampling on
Quantitative Detection
Small sub-sample
Large Sub sample
More particles
Better representation
Large DNA prep
Uniform results
Fewer particles
More variability
Easy DNA extraction
Higher Throughput
Variability in results
Challenges related to DNA Extraction and
Impurities at Low Level of GM Detection
Charge switch
0.5%
CTAB
0.5%
Endogenous control
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 switch extraction method
Challenges related to DNA Extraction and Impurities
at Low Level of GM Detection
PMU Extraction Kit: 5%, 2%, 1%, 0.5%, 0.1%
Issues Related to Quantification of
DNA for Quantitative PCR
Evaluatio #
DNA
n Type
Sample conc.
(ng/ul
OD (UV) 704
20.0
Min
(ug/ul)
Max
(ng/ul)
17.17
24.41
Fl (PG)
5632
5.14
0.00
11.41
Fl (QG)
5632
5.14
0.19
7.95
Influence of Initial DNA Conc.
CTAB
Qiagen
PMU
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
Strategies for Quantification of
Biotech Traits
 Designing primers from the promoter and
terminator sequences
 Gene specific
 Event specific
 Construct specific (used during the
transformation)
Examples of Some commercial
Events
 Event 176
Pepc promo
Cry1A(b)
T35S
a
Cry1A(b)
Pepc promo
b
35S
c
Bar
T35S
T35S
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
Examples of Some commercial
Events
Event Bt11
a
Bt 11 has two expression
Cassettes
Both cassettes contain the
35S promoter
b
Mon 810
Mon 810 has only
Only copy of promoter and gene
sequences
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
Most of commercial agricultural GM products can be detected by
using the sequences from 35S promoter and NOS terminator
Examples of Transgenic Events
with 35S Promoter Sequences
Event Name
non GM maize
Event176
Bt11
T25
Mon810
GA21
NK603
Mon802
MON863
TC1507
Non GM soy
GM soy (RR)
P35S
+
+
+
+
+
+
+
+
+
T-35S
+
+
?
?
-
NOS
+
?
+
+
+
?
?
+
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
Challenges in GM Quantification
Using 35S Promoter Sequences
Event Bt11
a
Bt 11 has two copy of 35S
Where as Mon 810 has single
Copy of 35S sequences
b
Mon 810
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
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.
Challenges in GM Quantification
with 35S Promoter Sequences
35S copy
number
1 copy
event
2 copy
event
Event
Trait
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
3 copy
0.3
0.15
Mon810+Mon863 ECB +CRW
Quantification of Stacked Trait with
35S
Mon 810 + NK603
Ct=28
Ct= 31
Mon 810
Ref Ct = 29
Designing Primers and Probe Specific to
Gene for Real Time PCR Assay
 Detection of Transgene
Bt gene
Bt11
Mon810
Event 176
 Design markers for specific gene
 CP4 gene for roundup
 Pat and Bar gene for Liberty
Design primers from
 Primer specific to Bt gene
The gene sequences
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
Challenges in Designing
Primer/Probe for Quantitative PCR
Example of cry 1A(b) gene present in three transgenic event
Different Copy Number of Gene for
Each Event
 Event 176
Pepc promo
Mon 810
T35S Pepc promo
T35S
35S
Bar
T35S
NK 603 has two
Copy of CP4
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
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
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
Insertion site/Event site
Corn Chromosome
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
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.
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
Thank you