Fig 1.
Droplets in the intermediate region.
The droplet rain between positive and negative droplets prevents a clear threshold setting. Thus, the different thresholds (low or high) result in a variation of approximately 10% of the positive droplets.
Table 1.
List of primers and probes used.
Fig 2.
Droplet pattern with increased concentrations of polymerase, primers, dNTPs and MgCl2.
ddPCR was performed with EGDe DNA (≈ 103copies/sample, Ch1 (+)) and ΔprfA DNA (≈ 103 copies/sample, Ch2 (+)) using modifications of the components Taq polymerase (+1.5 U, +0.75 U), MgCl2 (+25 nmol), dNTPs (+2.5 nmol each), Taq polymerase (+0.75 U) with MgCl2 (+12.5 nmol), Taq polymerase (+0.75 U) with MgCl2 (+12.5 nmol) and dNTPs (+2.5 nmol each) as well as increased primer concentrations (937.5 nM final concentration).
Fig 3.
Droplet cluster with digested DNA, different probe concentrations, dyes and quenchers.
ddPCR with EGDe DNA (≈ 103 copies/sample) shows that the rain is similar when DNA is digested with BamHI, higher or lower concentrations of the probe were applied, FAM (blue, Ch1 amplitude) or VIC (green, Ch2 amplitude) as dyes were used, MGB probes (all but “BHQ1”) or probes from another supplier were introduced. Unless indicated otherwise, 312.5 nM MGB probe with FAM as dye and the non-fluorescent quencher were used. For optimal comparison of quenchers and dyes, the last three probes were ordered and tested simultaneously.
Fig 4.
Influence of cycle number, elongation and denaturation times and ramp rate.
(a) With a higher number of cycles in the PCR, separation of the positive and negative droplets is more distinct and the fluorescence level of the positive droplets higher. (b) Lower ramp rate (2°C/s or 1°C/s) promotes better droplet separation as well as longer elongation or denaturation steps do (unless indicated otherwise, one minute elongation, 30 seconds denaturation and a ramp rate of 2°C/s was used). ddPCR was performed with EGDe DNA (≈ 103 copies/sample).
Fig 5.
Usage of different temperatures in the PCR program.
A gradient PCR between 50°C and 70°C (a) with EGDe DNA (≈ 103 copies/per sample) was performed and droplets subsequently analyzed with best results at 62.2°C and 57.7°C. For more precise determination of the temperature, a gradient PCR between 62°C and 55°C (b) was performed (two minutes elongation time) with best separation of the droplets using 59.4°C.
Fig 6.
Droplets in the rain region in the optimized ddPCR.
When run at 59°C, increased denaturation and elongation time (one and two minutes, respectively) and 60 cycles, the droplet rain between positive and negative droplets comprises below 1% of the positive droplets, depending on the threshold (high or low).