User manual

Droplet Digital
™
PCR Applications Guide | 27
Multiplexing with EvaGreen
Differences in droplet amplitude due to differences in amplicon length or optimal annealing
temperature allow for multiplexing in a single well. Figure 2.15 shows a 2-D fluorescence
plot containing two amplicons, where differences in amplitude are observed due to
differences in optimal annealing temperature. Primer set 1 has an optimal T
m
of 63˚C,
therefore droplets having this target have relatively high fluorescence amplitudes at 63˚C.
Primer set 2 has an optimal annealing/extension temperature of 59˚C and therefore has a
much lower fluorescence amplitude at 63˚C. Distinct separation of these clusters allows a
copy number to be calculated with high precision (Figure 2.15). Similarly, varying amplicon
length instead of PCR efficiency can be used to measure multiple targets in the same well.
However, when using amplicon length to multiplex with EvaGreen it is important to use an
optimal annealing/extension temperature for thermal cycling (Figure 2.13).
Fig. 2.15. Multiplex assays performed using ddPCR with EvaGreen. Differences in amplitude are due to
differences in optimal annealing temperature. T
m
, melting temperature.
Reference
Untergasser et al. (2007). Primer3Plus, an enhanced web interface to Primer3.
Nucleic Acids Res 35 (web server issue), W71–W74.
Designing Droplet Digital
™
PCR Experiments
3,000 4,000 5,000 6,000 7,000
Channel 2 amplitude
Channel 1 amplitude
Annealing/extension: 63˚C
Primer set 1: T
m
= 63˚C
Droplets with
amplicons
from both
primer sets
Primer set 2: T
m
= 59˚C
16,000
14,000
12,000
10,000
8,000
6,000
4,000