Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers

Duplex digital polymerase chain reaction — ddPCR — allows the simultaneous quantitative detection of two distinct genetic markers.

To begin, take a sample containing two different target sequences. Add a mixture containing a thermostable DNA polymerase enzyme, dNTPs, and target-specific primers. Next, add two different target-specific oligonucleotide probes, labeled with fluorescent reporters and a quencher molecule to detect amplified targets.

The solution is emulsified with oil to partition the target sequences into numerous nanodroplets. Each droplet containing the target acts as an individual reaction vessel.

Start the thermal cycling process for the amplification reaction inside the individual nanodroplets.

At a high denaturation temperature, the double-stranded targets separate into single strands.

Lower the temperature to reach the appropriate annealing temperature, allowing the primers and oligonucleotide probes to bind to the template.

The proximity of the quencher suppresses the fluorescence of the reporter. At the extension temperature, DNA polymerase extends the primers and cleaves the oligonucleotide probe. The unbound reporter emits fluorescence.

Pass the droplets through a droplet reader — a fluorescence-detection system. Detection of single or dual fluorescence indicates positive droplets containing targets, while the absence of a signal indicates negative droplets.

The ratio of positive droplets to the total number of partitions determines the initial concentration of the target sequences.