Digital PCR
Related: qPCR · PCR · Nucleic Acid Quantification
1. Principle
Digital PCR partitions a reaction into thousands of micro-reactions (droplets or chips) so each partition is scored positive/negative after PCR; absolute concentration is calculated from the Poisson distribution without a standard curve.
2. Step-by-Step Procedure (conceptual)
- Prepare mix similar to qPCR; partition (droplet generator or nanoplate).
- Thermocycle partitions.
- Read fluorescence per partition.
- Compute copies/µL from positive fraction (Poisson stats).
3. Interpretation
- Absolute quantification with high precision at low copy numbers.
- Excellent for rare mutant fractions (resistance SNPs) against wild-type background.
- Pitfalls: Rain/cluster gating; saturation at very high concentrations; cost/throughput.
4. Clinical / Reference Use Cases
- Reference quantification of standards; viral load discrepancy troubleshooting.
- Detection of low-frequency resistance alleles.
- Environmental/low-biomass pathogen detection research.
- Calibrator for qPCR assays.
5. Comparison with Other Methods
| Method | Quantification | Standard curve? |
|---|---|---|
| qPCR | Relative / std-curve absolute | Usually yes |
| Digital PCR | Absolute | No |
| Nucleic Acid Quantification dyes | Mass, not copies of a gene | N/A |
6. Mnemonic / Visual Aid
Partition → Count ON/OFF → Poisson — digitization of PCR.
Active Recall
- Why can dPCR skip a standard curve?
- When is dPCR better than qPCR for resistance mutations?
- What limits dPCR at very high template loads?