Encyclopedia of Experiments: Biological Techniques
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Transcript
Digital polymerase chain reaction — or dPCR — is useful for quantifying single nucleotide variants — or SNVs — a mutation where the substitution of a single nucleotide at a specific position in the genome creates two nucleotide variations or alleles.
To begin quantification using the chip-in-a-tube dPCR technique, take a DNA sample containing SNVs — the mutant allele, and the corresponding wild-type allele. Add a mixture containing a thermostable DNA polymerase enzyme, dNTPs, and primers.
Next, add allele-specific oligonucleotide probes — labeled with reporters of different-colored fluorescence to distinguish between the alleles — and a quencher molecule. The proximity of the quencher to the reporter suppresses its fluorescence.
Partition the solution into the reaction chambers of a microfluidic chip. Each chamber containing an allele serves as an independent reaction vessel.
Place the tube with the chip inside a thermal cycler, and start the reaction. At a high temperature, the double-stranded DNA denatures into single strands. Lower the temperature to anneal the primers and oligonucleotide probes to their complementary regions.
At an appropriate extension temperature, the DNA polymerase extends the primers and cleaves the probe. Distance from the quencher enables the reporter's fluorescence emission.
Read the different-colored fluorescence signals, and create a position plot to detect the chambers containing the alleles.
To determine the frequency of the mutant allele — the variant allele fraction — compute the ratio of chambers containing the mutant versus the wild-type allele.
