User manual

24 | Droplet Digital
™
PCR Applications Guide
Designing Droplet Digital
™
PCR Experiments
ddPCR Using the QX200 System and EvaGreen dsDNA Dye
The QX200 system can measure amplified DNA using Bio-Rad’s QX200 ddPCR EvaGreen
supermix, template, and a pair of primers. EvaGreen dsDNA binding dye is similar to
SYBR
®
Green in that it fluoresces upon binding double-stranded DNA (Figure 2.11).
Fig. 2.12. Accurate quantification of target is obtained using Bio-Rad’s QX200 system with the QX200
ddPCR EvaGreen supermix. A, threshold set above primer-dimer; B, accurate quantification of the target is
obtained without redesigning primers.
0 50,000 100,000 150,000 200,000 250,000
Event number
Sample
Channel 1 amplitude
Channel 1 concentration, copies/µl
30,000
20,000
10,000
300
250
200
150
100
50
0
A02
A02
55˚C65˚C 65˚C 55˚C
237
233
0.135 0.131 0 .19 6 0.261 0.126 0.261
0.0 641 0.0671
242
234
229
243
236
237
B02
B02
C02
C02
D02
D02
E02
E02
F02
F02
G02
G02
H02
H02
A04
A04
B04
B04
C04
C04
D04
D04
E04
E04
F04
F04
G04
G04
H04
H04
No template control No template controlSample
A B
Annealing
temperature
Primer-dimer
Primer-dimer frequency increases at
lower annealing temperatures and is
visible in the no template control wells.
Template Template
Fig. 2.11. EvaGreen dye binds to dsDNA via a “release-on-demand” mechanism.
Inactive form
of EvaGreen
Active form
of EvaGreen
DNA
EvaGreen-DNA complex
EvaGreen dsDNA binding dye enables double-stranded DNA detection with the
convenience and savings of only needing primers to amplify and detect product with
the added high-resolution features of ddPCR. ddPCR with EvaGreen can be used with
applications such as gene expression, copy number variation, DNA rearrangement
detection, micro RNA detection, and multiplexing.
The fluorescence amplitude of each droplet with EvaGreen varies with amplicon length
and with PCR efficiency. Longer amplicons will bind to more EvaGreen dye molecules and
therefore be brighter. PCR reactions that are not at their most efficient annealing/extension
temperature will result in fewer amplicons in each positive droplet at end point and therefore
lower fluorescence amplitude. Despite the range in positive fluorescence amplitude,
accurate quantification is still routinely achieved. These features provide the user the ability
to measure length or PCR efficiency as well as the ability to measure multiple targets in the
same well while only using primers. Additionally, primer-dimers and off-target amplicons can
be detected as low fluorescence amplitude droplets. With the EvaGreen supermix reactions
it is possible to simply set the threshold above the primer-dimer in order to obtain an
accurate quantification of the target without having to redesign the primers (Figure 2.12).