LGC eBook - 2017 - 10

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

in combination with the flexibility of
TOCE technology, contributes to exceptional ease of use in design and development of TOCE assays. This further
distinguishes TOCE from other technologies that require highly sophisticated
bioinformatics and extensive target
information to provide specificity for
a limited number of targets.
TOCE technology also allows for mutation detection to discriminate even one
base difference accurately, enabling detection of clustered point mutations. In
Figure 2, a signal is generated only when
a mutant-specific Pitcher binds to a mutated target sequence, discriminating
mutations from a wild-type sequence.
High Multiplicity in a
Single Channel
TOCE technology enables identification
of multiple target analytes simultaneously in a single channel. The signal
can be measured in real time and/or
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analyzed by Catcher melting
temperature (Tm) to detect the
presence of the target analyte.
Figure 3 shows a representative
example of Pitcher-Catcher designs
to differentiate between three unique
melt curves for three targets in a single
channel, with Tm values ranging from
60°C, 65°C, and 70°C for the three
unique targets, respectively.
Unique, target-specific Tm profiles
can be controlled by the engineered
sequence of TOCE Catcher oligos, and
by designing unique, paired complementarity of each Catcher to one and
only one target-specific Pitcher.
Another feature of TOCE is that the
Catcher is a fluorescently labeled artificial template, and therefore the Tm
can be controlled by adjusting the
sequence design parameters of the
Catcher oligo. For TOCE assay optimization, the Tm can be easily adjusted and
is not limited by the target sequence.
The controlled Tm of the Catcher

Figure 2. Allelic detection and discrimination enabled by
mutant-specific Pitcher-Catcher pairs.

Figure 3. Controlled Tm profiles through designed sequence
variations of the Catcher.

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LGC eBook - 2017

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