Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants

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 pol