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