User manual

12 | Droplet Digital
™
PCR Applications Guide
When designing primers for a target sequence, follow these guidelines:
■
Design primers that have a GC content of 50–60%
■
Strive for a T
m
between 50 and 65°C. One way to calculate T
m
values is by using the
nearest-neighbor method. Use the T
m
calculator at http://www.basic.northwestern.edu/
biotools/oligocalc.html, with values of 50 mM for salt concentration and 300 nM for
oligonucleotide concentration
■
Avoid secondary structure and adjust primer locations so they are outside the target
sequence secondary structure, if required
■
Avoid repeats of Gs or Cs longer than 3 bases
■
Place Gs and Cs at the 3' nucleotide of primers when possible
■
Check forward and reverse primer sequences to ensure no 3' complementarity
(avoid primer-dimers)
Designing Probes
The QX100
™
Droplet Digital PCR system is compatible only with TaqMan hydrolysis probes.
The QX200 system is compatible with TaqMan hydrolysis probes and EvaGreen double-
stranded DNA (dsDNA) binding dye. Using EvaGreen or SYBR
®
Green on the QX100 will
damage the system.
Neither the QX100 nor the QX200 system is compatible with SYBR
®
Green. Advantages of
using hydrolysis probes include high specificity, a high signal-to-noise ratio, and the ability
to perform multiplex reactions. Hydrolysis assays include a sequence-specific, fluorescently
labeled oligonucleotide probe in addition to the sequence-specific primers. TaqMan assays
exploit the 5' exonuclease activity of certain thermostable polymerases. The hydrolysis
probe is labeled with a fluorescent reporter at the 5' end and a quencher at the 3' end.
When the probe is intact, the fluorescence of the reporter is quenched due to its proximity
to the quencher (Figure 2.1). The amplification reaction includes a combined annealing/
extension step during which the probe hybridizes to the target and the dsDNA-specific
5' to 3' exonuclease activity of Taq or Tth cleaves off the reporter. As a result, the reporter
is separated from the quencher, resulting in a fluorescence signal that is proportional to the
amount of amplified product in the sample.
Designing Droplet Digital
™
PCR Experiments