Chip-Based Digital PCR to Detect Rare Transcript Variants Using a Nanofluidic Chip

The chip-based digital PCR technique partitions a single reaction into the chambers of a nanofluidic chip. The amplification of the partitioned sequences enables the detection of rare transcript variants — non-canonical transcripts occurring at a low frequency.

To begin, take a sample containing cDNA of the target rare transcript variant and the common variant. Add a solution containing thermostable DNA polymerase, dNTPs, and primers.

Add common and rare transcript variant-specific oligonucleotide probes — labeled with different fluorescent reporters and quencher molecules. The quencher absorbs the fluorescence of the reporter when in close proximity.

Load the mixture onto the nanofluidic chip. The solution is partitioned into uniformly-sized nano-chambers. Each chamber containing a cDNA serves as an independent reaction vessel.

Start the thermal cycling process. A high denaturing temperature separates the double-stranded DNA into single strands. Lower the temperature, allowing the primers and oligonucleotide probes to anneal to the complementary regions on the single DNA strands.

Increase the temperature to reach the extension step, where the DNA polymerase extends the primer and cleaves the probe. Distance from the quencher enables the reporter's fluorescence emission. Read the fluorescence signal.

Create a scatter plot depicting the chambers with the amplified rare-transcript target and the chambers devoid of the target. Positive signals for the target indicate the presence of the rare-transcript variant in the sample.

To begin, let the master mix and the assay thaw at room temperature for at least 20 minutes. Then, load 0.5-milliliter tubes with 6-microliter aliquots of the cDNA samples. Also, make a no-template control tube. Next, gently vortex the master mix and aliquot 8.7 microliters per reaction into a sterile tube.

Then, for each reaction, add 0.87 microliters of the custom assay primer, and add 1.83 microliters of nuclease-free water. Mix the combination gently, and transfer 11.4 microliters to each tube containing cDNA, and to the no template control. Then, mix the tubes gently and pool the tube contents using pulse centrifugation. For optimal results, load the chips as soon as possible.

Plug in the chip loader, and wait until the indicator light turns green. Then, prepare the immersion fluid syringe by gently pulling the plunger to 1 to 2 millimeters, then, remove the cap, and add the tip.

Next, take note of the code written on the lid of a new chip, then, hold the lid carefully by the side, peel away the protective film, and position it in the lid nest with the sticky side up. Now, press down the lever to open the clamp, and carefully load the chip into the chip nest.

Next, put a new loading blade into the loader. Push it gently to ensure that it is firmly in place. Now, transfer 14.5 microliters of reaction mix onto the loading blade. Do not make air bubbles, and do not deflect the blade. Then, press the black "Loading" button to distribute the volume onto the chip.

It is important to confirm the loading blade is in place. Then, while filling the blade, do not depress the pipette to the second stop to avoid introducing bubbles. Also, do not deflect the blade with the tip.

Proceed by using the syringe to transfer about 20 drops of immersion fluid onto the chip surface. Take care not to touch the surface with the tip, and completely cover the surface without overflowing it. Next, rotate the rotor arm to make the lid come into contact with the chip, and press down for 15 seconds. Then, press the lid button to release the chip and return the loader arm to its resting position. Now, open the clamp, and hold the assembled chip at a 45-degree angle to carefully dispense the immersion fluid through the fill port via a syringe. Then, rotate the chip slightly to remove air bubbles, and then, remove any excess fluid with the sterile wipe.

Avoid spilling immersion fluid on the border of the tip. If that happens, carefully remove excess before sealing.

Finally, seal the chip case by gently peeling away the label on the lid, and then, blocking the fill port for at least 5 seconds. Now, use the chip within two hours, and until then, store it in the dark.

To set up the reaction, open the lid and install the adapters on both blocks, even when a single block is used. Then, place a chip on the sample blocks and orient its fill ports towards the front of the thermal cycler and elevate the port slightly. Use empty chips to balance the two blocks.

Next, lay the thermal pad over the setup to completely cover the chips. Then, program the cycles, close the lid, and start the reaction.

When the reaction is over, turn off the thermal cycler, remove the thermal pads, and then, remove the chips. Let the chips thaw to room temperature for at least 10 minutes in the dark and analyze them within an hour. Clean the chip surface with isopropanol while inspecting them for leaks or other issues.

To save the data, insert a USB memory stick into the detector system where the data can be saved. Then, open the chip tray of the detector system and load the chip face up. Close the tray and wait 30 seconds for the data to be processed. Then, remove the chip and repeat the process for the next chip.

Next, move the USB stick from the detector to a computer, and transfer the files. Open the cloud-based software, then, create a project and import all the data files for the chips of interest. Then, under "Defined Chips" tab, select the dye and assay used. Determine if the chip is acceptable by visualizing it in the "Review Data" tab. If the sample was loaded well, at least 13,000 points should be usable.

Next, look at the scatterplot for the selected chip. There, apply a threshold defined by the assay type. For fluorescein amidite reporter dye signal, use a threshold of 6,000. Now, remove any dubious signals that could result in false positives. Select the questionable spot on the scatterplot using the "Lasso" tool and select the "Undetermined" option.

All the remaining positive spots indicate the presence of cDNA copies of the rare target analyzed.