Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes
Transcrição
Digital droplet PCR detects indel mutations — random insertion or deletion of nucleotides in the DNA. This is achieved by partitioning the sample into tiny droplets and independently amplifying the DNA in each droplet.
To perform ddPCR, take a DNA sample containing wild-type and mutant variations of the target gene. Load the sample mix and oil into distinct compartments of the droplet generator chamber containing the microfluidic channel.
The sample and oil flow rapidly through the narrow channels, emulsify at the junction, and are partitioned into several droplets, each containing a few DNA molecules.
Now, take a PCR plate with the buffer containing forward and reverse primers, dNTPs, thermostable DNA polymerase, and two probes tagged with different fluorophores and quencher molecules. Each probe is specific to a particular type of allele — wild-type or mutant one.
Transfer the droplets to the PCR plate. Place the plate in a thermocycler, and run the reaction. During denaturation, the DNA strands separate. At annealing temperature, the primers and probes anneal to the template DNA.
The probe with green and orange fluorophores binds to the wild-type and mutant DNA sequence respectively. Later, DNA polymerase extends the primers, cleaving the fluorophore and quencher molecules from the probe, generating a fluorescence signal.
Analyze the amplified product. The droplets showing green fluorescence contain the wild-type DNA sequence, while the orange fluorescence indicates the indel mutation.
