Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

This method can be used as a diagnostic tool for cancer to analyze the circulating tumor DNA. This technique interrogates multiple genetic alteration in regions of cluster mutations in a single reaction. This helps to save precious patient samples.

This method can provide insight to tumor burden on its mutational profile. It can now also be applied to a molecular analysis as an alternative to qPCR to obtain an absolute quantification of nucleic acid target sequences. Collect blood samples in seven-milliliter EDTA tubes.

Two tubes are recommended to collect around four milliliters of plasma. Centrifuge the blood samples for 10 minutes at 820 times gravity within three hours of the blood draw to separate red blood cells, white blood cells, and plasma. The time between the sampling and the centrifugation is critical to obtain high-grade, T-cell-free DNA not contaminated with DNA released from white blood cells.

Following centrifugation, use a serological pipette to collect the supernatant without touching the white blood cell layer. Around two milliliters of plasma are usually recovered per EDTA tube. After transferring the plasms to two milliliter tubes, centrifuge the aliquots at 16, 000 times gravity for 10 minutes at 15 degrees Celsius to remove cell debris.

Carefully collect the plasma without disturbing the pellet and transfer to two milliliter cryogenic tubes. Store at negative 80 degrees Celsius until needed. Begin by adding 400 microliters of proteinase K to a 50-milliliter tube.

Then, add four milliliters of plasma and 3.2 milliliters of ACL lysis buffer containing one microgram of carrier RNA from the extraction kit. Close the tube and mix by vortexing for 30 seconds to obtain a homogenous solution. Then, incubate at 60 degrees Celsius for 30 minutes.

After the incubation, add 7.2 milliliters of buffer ACB to the lysate to optimize the binding of the circulating nucleic acids to the silica membrane. Mix by vortexing for 15 to 30 seconds. Then, incubate the tube on ice for five minutes.

Attach the column and the 20 milliliter extender onto the vacuum pump. Close the unused portions to allow vacuum aspiration. After briefly centrifuging the tube, carefully introduce the mixture into the tube extender.

Then switch on the vacuum pump and allow the lysate to be drawn through the columns completely. Remove and discard the tube extender. Next, apply 600 microliters of buffer ACW1 to the column.

When buffer ACW1 has drawn through the column, add 750 microliters of ACW2 buffer, and finally, 750 microliters of 96 to 100%ethanol. Then, place the column in a clean two-milliliter collection tube and centrifuge at full speed for three minutes to eliminate ethanol. After placing the column into a new two milliliter collection tube, open the lid and then incubate at 56 degrees Celsius for 10 minutes to dry the membrane completely.

After the incubation, place the column into a clean 1.5 milliliter low-binding tube. Carefully apply 36 microliters of RNase-free water with 0.04%sodium azide. Then, close the lid and incubate at room temperature for three minutes.

Centrifuge at full speed again for one minute to elute the nucleic acids. Then, store at negative 20 degrees Celsius until needed. Begin by thawing and equilibrating the reaction components to room temperature in a clean PCR hood.

Then prepare a 20 times mix of primers and probes in RNase and Dnase-free distilled water with each primer at 18 micromolar concentration and each probe at five micromolar concentration. For all individual PCR reactions, prepare a sample mix by combining 10 microliters of two times ddPCR Supermix, one microliter of 20 times primers and probes mix, and up to 10 nanograms of the DNA sample in 20 microliters of distilled water. Vortex the reaction mixture thoroughly to ensure homogeneity.

And briefly centrifuge to collect contents at the bottom of the tube before dispensing. Insert the ddPCR cartridge into the holder and load 20 microliters of sample reaction mix into the middle wells of the cartridge for droplet generation. Add 70 microliters of droplet generator oil into the bottom wells.

Insert the cartridge with a gasket into the droplet generator. Droplets will be produced in the top wells of the cartridge. Slowly and gently aspirate 40 microliters of the droplets from the cartridges and dispense into a 96-well PCR plate.

Seal the final PCR plate with aluminum foil using a plate sealer immediately after transferring droplets to avoid evaporation. Briefly centrifuge the plate to collect contents at the bottom of the wells. Run a conventional PCR amplification on a thermal cycler.

When the run is completed, briefly centrifuge the plate to collect contents at the bottom of the wells. After PCR amplification, use the droplet reader to count PCR positive and PCR negative droplets following the manufacturer's instructions. The following are representative results obtained during optimization steps of the drop-off ddPCR assay.

This image shows detection of mutant DNA and wild-type DNA in a reaction containing 100%mutant DNA, 5%mutant DNA, and 100%wild-type DNA. This image shows examples of plasma samples analyzed with the KRAS and EGFR drop-off ddPCR assays showing the detection of single nucleotide and multiple substitutions in exon two of KRAS and a deletion in EGFR exon 19. While attempting this procedure, it's important to proceed carefully at each step with a special attention at the droplet generation, which is a critical step of this method.

After its development, this technique paved the way for researcher and clinician in the field of cancer research to optimize tumor mutation analysis using liquid biopsy.