End-point zygosity and CNV determination based on post PCR Rn/ΔRn reading is expected to reduce the cost and increase throughput as compared to current real time Cq-based measurement. There is an immediate need by AgBio customers to screen seed zygosity and copy number of transgenes through end-point reading. Moreover, Agbio customers expect to determine zygosity and CNV using crude samples in order to stream-line their workflow. For endpoint quantification, the major challenge is to control the saturation of PCR to maintain the segregation of end-point intensity according to the input copy number of the target transgenes in reference to a control gene. We have tried a few approaches such as Asymmetric PCR, ARCS (Amplification Ratio Control System) PCR, Dual Tailing PCR, pyrophosphate removal and controlling plateau of PCR by running a third PCR in the background. Among the tested approaches, controlled plateau of PCR (CoP’ed PCR) showed best separation of copy number through end-point PCR reading. We further optimized CoP’ed PCR conditions using purified DNA and crude plant and blood samples. By CoP’ed PCR, we are able to do zygosity with crude plant samples to satisfy the immediate needs for Agbio customers. Furthermore, since this approach improves the sensitivity for the copy number separation for not only end-point but also real-time PCR for crude blood samples, in the future, it can be used for CNV analysis directly from human blood.

1. A Thermo Fisher Scientific Brand
End-point zygosity and CNV determination from crude samples
or CoP’ed PCR
© 2014 Thermo Fisher Scientific Inc. All rights reserved. For RUO only.
64th ASHG PB# 588T
Chunmei Liu, Shoulian Dong and Junko Stevens
Life Technologies, 180 Oyster Point Blvd. , South San Francisco, CA 94080, USA
ABSTRACT
End-point zygosity and CNV determination based on
post PCR Rn/ΔRn reading is expected to reduce the
cost and increase throughput as compared to current
real time Cq-based measurement. There is an
immediate need by AgBio customers to screen
seed zygosity and copy number of transgenes
through end-point reading. Moreover, Agbio customers
expect to determine zygosity and CNV using crude
samples in order to stream-line their workflow. For end-point
quantification, the major challenge is to control
the saturation of PCR to maintain the segregation of
end-point intensity according to the input copy number
of the target transgenes in reference to a control gene.
We have tried a few approaches such as Asymmetric
PCR, ARCS (Amplification Ratio Control System)
PCR, Dual Tailing PCR, pyrophosphate removal and
controlling plateau of PCR by running a third PCR in
the background. Among the tested approaches,
controlled plateau of PCR (CoP’ed PCR) showed best
separation of copy number through end-point PCR
reading. We further optimized CoP’ed PCR conditions
using purified DNA and crude plant and blood samples.
By CoP’ed PCR, we are able to do zygosity with crude
plant samples to satisfy the immediate needs for Agbio
customers. Furthermore, since this approach improves
the sensitivity for the copy number separation for not
only end-point but also real-time PCR for crude blood
samples, in the future, it can be used for CNV analysis
directly from human blood.
INTRODUCTION
Zygosity information is always required for effective
breeding and colony maintenance of the transgene,
therefore, estimating transgene copy number is critical
to the characterization and selection of candidate
transgenic plants. Current qPCR based copy number
determination relies on cycle of threshold (Ct/ΔCt),
which requires real time instruments. In order to
increase the throughput, reduce cost and increase turn
around time, there is an immediate need from AgBio
customers to do copy number determination using
end-point readings (Rn/ΔRn, end-point Zygosity). The
major challenge for the end-point Zygosity is the
saturation control of PCR. In addition, since
customers will use crude samples to streamline their
workflow, duplexed assays are needed to quantitate
the level of transgene relative to an endogenous
normalization gene and the saturation control for
duplex assays has to be in the same manner. We have
tested different approaches to achieve the end-point
Zygosity.
3,2,1
Ct Based Copy# Rn Based Copy#
APPLICATIONS
By control end-point PCR saturation, we provide
customers a solution to do end-point Zygosity, which
helps to increase throughput and reduce cost. In the
future, it can also be used for blood chromosome copy
number detection.
Figure 4. CoP’ed PCR Improves End-point
Zygosity- Human Blood Crude Lysate
CoP’ed PCR increased copy number separation for both
real time and end-point Zygosity: blood crude lysates from
different individuals was used for PCR to evaluate the X
chromosome copy#. Without input normalization, inclusion of
background PCR, we has clear separation of X chromosome
copies between female and male not only for end-point but
also for real time.
CONCLUSIONS
We have tried 6 different approaches to control
PCR saturation at end-point to provide customer a
solution to do end-point Zygosity. Among all the
approaches we tried, A, B, C showed much lower
PCR efficiency in the exponential
phase ,therefore ,performance of the real time Ct
based copy number separation was affected. Our
goal is to achieve end-point Zygosity without
sacrificing performance for the real time. The
assumption of the D approach is to prevent
depletion of Taq enzyme caused PCR saturation.
Data showed that this approach also led to very
low PCR amplification efficiency. Assumption of
approach E is that PCR saturation was caused by
accumulation of Ppi, therefore, by adding
pyrophosphatse in the PCR , we can control the
saturation. Unfortunately, in our experiment, we
did not see any effect. The last approach (F) is to
run 3rd PCR in the background to slow down the
saturation of target PCR. After optimization of
background PCR (CoP’ed PCR), we are able to
achieve end-point Zygosity without impacting real
time performance using purified gDNA aneuploidy
samples. To simplify customer workflow, we did
further testing using crude lysate from plant (corn)
leaves as well as human blood samples. Our data
shows that by running the background PCR
(CoP’ed PCR), we are able to not only achieve
end-point segregation , but also improve the real
time separation in blood samples. In the future,
there could be applications for human copy
number aberration detection from blood with
increased sensitivity.
REFERENCES
1. Pierce, K.E. ,Sanchez, J.A. & Wangh, L.J. (2005)
Proc.Natl.Acad.Sci. USA102, 8609-8614
2. Guthrie, P.A.I, Gaunt,T.R. & Day, L.N.M (2011) Nucleic
Acids Research 1-12
3. Ingham, D.J., Beer, S.& Hansen, G (2001) BioTechniques
31: 132-140
ACKNOWLEDGEMENTS
We want to thank all the people who helped and
supported this project, including Allen Nguyen, Mark
Shannon, Kelly Li , Janice Au-Young .Special thanks was
given to Caifu Chen for his support .
Strategies to Control PCR Saturation
RESULTS
Figure 1. STD Approach: Chr21 Assays with Different
Amount gDNA (100X)
Unsupervised cluster based on Delta Rn Ratio CNV/RnaseP:purified
gDNA with varying input cross100 fold were tested using aneuploidy
samples. ΔRn ratio of target assays and normalization assay was used
to do hierarchical clustering. Ct cutoff for Rn here is 30. Within the
dynamic range (no saturation), we are able to segregate 1 ,2 and 3
copies with sample input range of 100 fold (100-10,000 copies).
Figure 2. CoP’ed PCR Reduces Plateau of Target
PCR-gDNA
Control of PCR saturation through CoP’ed PCR : two different
assays are tested with fixed amount of purified gDNA. At the end of
PCR(Ct 40) , we are able to distinguish 1,2 and 3 copies with the
help of running of background PCR. No normalization is needed as
sample input is the same .
Figure 3. CoP’ed PCR Reduces Plateau of Target PCR
-Corn Crude Lysate
CoP’ed PCR also control the PCR saturation for crude sample:
with the spike-in of corn leave crude lysate into gDNA samples,
there is much better separation of different copies at the end of
PCR compared with control which all clustered together at the end.