Multiplex Droplet Polymerase Chain Reaction to Detect DNA Methylation in Leukocytes

DNA cytosine residue methylation is a characteristic feature of leukocytes or white blood cells.

Multiplex droplet polymerase chain reaction, or mdPCR, allows the simultaneous identification of multiple methylated DNA regions within the leukocytes.

Prepare a mix containing reaction buffer, DNA polymerase, and nuclease-free water. Add forward and reverse primers and fluorescent hydrolysis probes specific for each target region. The probes comprise differently-colored fluorophore reporters bound to quenchers to inhibit fluorescence emission.

Add bisulfite-converted DNA, wherein the unmethylated cytosines are deaminated into uracils while the methylated cytosines remain unchanged. Mix.

Fill a glass syringe with a carrier oil-surfactant mix and another with a carrier oil-PCR mix, and connect them to the microfluidic droplet generator. The carrier oil-aqueous PCR mix emulsifies into sub-nanoliter-sized droplets. The surfactant stabilizes the emulsion and prevents droplet merging.

Collect the droplets, and perform PCR. Within each droplet, the high denaturing temperature denatures double-stranded DNA into single-stranded DNA and activates the polymerase. The reaction temperature is lower