205- Amplify the sample to end point with a gradient enabled PCR
machine using the following cycling conditions (see Note 14):
3 min of initial denaturation at 95 °C, followed by 40 cycles of
10 s denaturation at 95 °C, 10 s of annealing at said optimized
temperature, and 20 s of extension at 72 °C. Then, a final
extension at 72 °C for 10 min and infinite holding at 12 °C
(see Note 15). - Transfer the PCR plate containing the amplicons onto a drop-
let reader where target DNA molecules will be aligned and
singulated through a detection channel within the system to
calculate the number of targets in an absolute manner accord-
ing to fluorescence amplitude. - Analysis can then be performed with the given software that
comes with the system such as QuantaSoft software version
1.7.4. - Assign threshold manually for each target as shown in Fig. 3
according to manufacturer’s protocol. - Then, choose the temperature that gives the most distinct
separation between the four clusters (as seen in Fig. 3 ) (see
Note 16). - In order to distinguish the clusters between one another, the
primer concentration of each target has to be manipulated. - Target 1 primer concentration can be tested at a range of
25–50 nM while Target 2 primer concentration can be tested
at a range of 100–150 nM. - Repeat steps 2– 9 of Subheading 3.4. Instead of using a gradi-
ent temperature, use the optimized temperature. - Primer combination that gives the most distinct separation
between the four clusters (as seen in Fig. 3 ) is chosen (see
Note 16). - In order to identify the different clusters, a single plex assay
containing Target 1 and Target 2 will be prepared separately.
The assay will run on another round of ddPCR in parallel with
the optimized duplex assay. - Three reactions of a single sample will be prepared to contain
primer set for only target 1, containing primer set for only tar-
get 2 and containing primer set for both targets. - The resulting 1D plot image should look like Fig. 4.
- Once the targets are identified, the assay can be run at 96 reac-
tions per run.
3.5 ddPCR Primer
Concentration
Optimization
3.6 Target Cluster
Determination
DNA Copy-Number in Esophageal Adenocarcinoma