Encyclopedia of Experiments: Biological Techniques
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Transcript
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.
