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.