How to interpret Drop-Off digital PCR assays using Crystal Miner
Digital PCR enables the Mutant Allelic Fraction (MAF) of a drop-off mutant to be quantified with high sensitivity using only two detection channels:
- A 1st channel to detect the Drop-Off Probe that spans the mutation site but is uniquely complementary to the wild-type sequence (e.g. “Blue Detection Channel” on the vertical axis in Fig. 1)
- A 2nd channel to detect the Reference Probe that hybridizes adjacent to the mutation site and is complementary to both the mutant and wild-type alleles (e.g. “Green Detection Channel” on the horizontal axis in Fig. 1)
In the presence of a wild-type allele, both the drop-off and reference probes will hybridize with their target, leading to a double positive signal (turquoise population in Fig. 1). Whereas in the presence of a mutant allele, even a single nucleotide mutation is enough to destabilize the hybridization of the drop-off probe so that only the reference probe anneals to its target leading to a single positive signal (green population in Fig. 1).
Why calculate a LOB in digital PCR?
In digital PCR (dPCR) false-positive events can arise from several sources of molecular biology noise. Determining a false positive cutoff when quantifying nucleic acids is therefore critical to the robustness of a dPCR assay. To define this cutoff, detection thresholds are commonly set above the Limit of Blank (LOB) value that must first be determined for each target during assay calibration.
We recommend the application of this LOB decision tree as a good practice before any new assay, particularly when a target is expected in low concentrations. Furthermore, the experimental LOB value is necessary both to determine if a reaction is positive or negative and to calculate the theoretical Limit of Detection (LOD).