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 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.

Add previously-designed primers, probes, and genomic DNA to a new 8-tube strip, to achieve a total volume of 15 microliters. Pipette up and down to mix.

Add the loading platform onto the chips built in the fresh 8-tube strip, and then, place the tube strip in the auto-loader. Make sure that there is a contact between the chips and the loading platform. Next, place the loading slider in the platform, and use a stopper to hold the slider off of the loader. Pipette 15 microliters of the PCR mixture near the tip of the slider, and then, press the loader button to run the loader for 1 minute.

Remove the tube strip from the loader after the run, and place it in sealing enhancer. Carefully push the slide lid and the edge of the top lid. Run the sealing enhancer for approximately 2 minutes. If sealing is incomplete, indicated by a puddle of liquid, repeat the run for an additional minute.

Add 230 microliters of sealing fluid to the tubes. Place the tube strip in the thermal cycler, and run the PCR as described in the protocol. If there is an uneven distribution of positive partitions, adjust the temperature or the duration of the PCR.

To detect and analyze fluorescence intensity of the PCR products, place the tube strip on the detection jig, and add 6 milliliters of distilled water. Remove any visible air bubbles using a pipette tip.

Load the jig into the detector. In the detection software, select "Fluorescence," "Experiment," and then "Sample/NTC" tabs, and click the "Run" button to start the run. After the complete run, confirm the position plot, histogram, and 2D scatterplot.

To collect the PCR product, remove the sealing fluid from the tube. Add 100 microliters of TE buffer, and vortex vigorously for 30 seconds. Briefly centrifuge the tubes in a tabletop centrifuge, and proceed as described in the text protocol, to finish collecting the PCR product.