This article discusses the use of multiplex droplet polymerase chain reaction (mdPCR) to identify methylated DNA regions in leukocytes. The method allows for the simultaneous analysis of multiple targets, enhancing the understanding of DNA methylation patterns.
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 lowered, allowing site-specific annealing of the primers and probes.
As the polymerase binds and extends the DNA-primer duplex, uracils amplify as thymines, while methylated cytosines amplify as cytosines, allowing the distinction of methylated and unmethylated regions. The polymerase further cleaves and releases the reporter from the probe, causing fluorescence emission.
Post-PCR, visualize the drops under a microscope. The fluorescence color indicates the presence of specific methylated DNA targets.
For droplet generation, mix 1 microliter of bisulfite-converted DNA with freshly-prepared probe master mix in a PCR tube, and collect the sample with a brief centrifugation. Use PEEK fittings to connect disposable fluidic tubing to two 250-microliter volume precision glass syringes, and pre-fill one precision glass syringe with 250 microliters of carrier oil containing 5% fluoro-surfactant, and one precision glass syringe with 50 microliters of carrier oil.
When both of the syringes have been loaded, load 100 microliters of the PCR mix into the syringe of carrier oil, and place a droplet microfluidic device onto the stage of an upright light microscope equipped with a high-speed camera.
For the observation and recording of droplet formation in real time, place the pre-filled syringes onto a programmable syringe pump, and use PEEK union with fittings to connect the tubing of the syringes to the tubing of the respective inlet channels of the droplet microfluidic device.
Place the tubing from the outlet of the droplet generator to the inside of a 0.5-milliliter PCR tube, and adjust the syringe pump flow rate to 2 microliters per minute. Do allow the droplet size to stabilize before collecting the resulting emulsion. Slowly and carefully collect the emulsion from the top of the tube, and transfer 75 microliters of the solution to a 200-microliter PCR tube for thermal cycling.
Then, confirm that the oil content in the PCR tube closely matches the volume of the dispersed phase to prevent coalescence of the droplets during thermal cycling, and place the 200-microliter tube into the thermal cycler.
繁體中文
