User manual

8 | Droplet Digital
PCR Applications Guide
Droplet Digital
PCR
Fig. 1.12. Estimating target concentration by ddPCR.
ddPCR is an end-point measurement that enables you to quantify nucleic acids without
the use of standard curves and independent of reaction efficiency. The present-or-absent
digital format (Figure 1.12) and ability to draw clear thresholds between positive and
negative droplet clusters for ddPCR means we now operate in a regime of absolute DNA
quantification without the need for external calibrators or endogenous controls. The region
separating these clusters is clear, such that the concentration output is not significantly
affected by the position of the threshold and results are not biased by the small fraction
of droplets that do not reach end point. This is another advantage of conducting many
thousand PCR reactions/20 μl sample (Figure 1.13).
0 0.2 0.4 0.6 0.8 1
Fraction of positive droplets, p (assumes 20,000 droplets/reaction)
Target copies/droplet
Copies of target/20 µl sample
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
100,000
90,000
80,000
70,000
60,000
50,000
40,000
30,000
20,000
10,000
0
0 positive
143 total
p =
No
targets
Sample 1
p =
6
143
Low
concentration
Poisson corrected
6.2/143
Sample 2
p =
34
143
Medium
concentration
Poisson corrected
38/143
Sample 3
p =
70
143
High
concentration
Poisson corrected
96/143
Sample 4