LGC eBook - 2017 - 9

Accelerating Science Using Integrated PCR Tools * High-Multiplex Real-Time PCR Design

(DPO™) primer pairs, "Pitchers" and
"Catchers." The DPO is Seegene's targetspecific primer design and provides highly
specific amplification of the target region.
The Pitcher is a tagging oligonucleotide
that hybridizes specifically to the target
region. The Catcher is a fluorescently
labeled artificial template.
Enhanced Target Specificity
DPO primers are an essential component
for optimizing target specificity of the
TOCE assay. They function normally in PCR,
but increase specificity of target amplification while minimizing nontarget interactions and primer-based artifacts. These
attributes are particularly important in a
high-multiplex TOCE assay as a means of
preventing spurious oligonucleotide
interactions that can generate nonspecific
signaling from the Catcher, or interfere
with the interaction between the Catcher
and the tagging portion of the Pitcher.
Ultimately, the use of DPO primers,
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Figure 1. Principles of TOCE
assay design and signal
generation during realtime PCR. (A) TOCE assay is
initiated with hybridization
of upstream and downstream
primers (DPO) and Pitcher to
the selected target sequence.
(B) Taq polymerase-mediated
extension of primers. (C)
Taq polymerase having a 5'
nuclease activity encounters
the target-bound Pitcher, and
cleaves the Pitcher, releasing
the tagging portion. The
sequence of released tagging
portion is complementary
to capturing portion of the
Catcher. (D) As the tagging
portion is fully extended
on the Catcher to create the
duplex Catcher, quenching is
diminished and the fluorescent
signal can be detected.

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LGC eBook - 2017

Table of Contents for the Digital Edition of LGC eBook - 2017