User manual

Droplet Digital
™
PCR Applications Guide | 13
Designing Droplet Digital
™
PCR Experiments
Fig. 2.1. In TaqMan assays, hydrolysis probes are hydrolyzed by Taq polymerase.
R Q
During annealing, the hydrolysis probe binds
to the target sequence
During extension, the probe is partially
displaced and the reporter is cleaved.
The free reporter fluoresces
Extension
R
Q
R
Q
R
Reporter
Q
Quencher
When designing probes, use the following guidelines:
■
The probe sequence must be chosen between the two primers of the amplicon. Primer
sequences cannot overlap with the probe, though they can sit directly next to one another
■
The T
m
of a hydrolysis probe should be 3–10°C higher than that of the primers
■
Choose a sequence within the target that has a GC content of 30–80%, and design the probe
to anneal to the strand that has more Gs than Cs (so the probe contains more Cs than Gs)
■
The probe should be <30 nucleotides long because distance between fluorophore
and quencher affects baseline signal intensity. Longer probes or T
m
enhancers are
recommended if needed to achieve the required T
m
■
Probes should not have a G at the 5' end because this quenches the fluorescence
signal even after hydrolysis
■
Black Hole Quencher or other nonfluorescent quenchers are recommended
■
T
m
enhancers for probes are recommended for single nucleotide polymorphism (SNP)
and rare mutation detection assays in order to keep the background fluorescence to
a minimum. Shorter probes discriminate better between single base differences in the
target amplicon(s)
The QX100 and QX200 systems are compatible with FAM and HEX or VIC dyes as well as
duplex analysis of FAM/HEX or FAM/VIC. Using dye combinations in duplex assays enables a
multiplex experiment and the ability to quantify and/or detect up to two targets per sample.
Designing an Assay
Because manual balancing of all the constraints for primer and probe designs is challenging,
time consuming, and error prone, we recommend software tools to ensure that all design
constraints are satisfactorily followed. One widely used tool for PCR assay design is Primer3
(Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology [MIT]).
Building on this software, the easy-to-use Primer3Plus web interface (Untergasser et al.
2007) makes the process of assay design even easier (Figure 2.2).