Similar to qPCR, ddPCR first generates multiple copies of the target mutation by polymerase chain reaction and using probes with fluorescence or another marker. However, ddPCR differs from qPCR in the way it quantifies. qPCR uses plastic wells or tubes, which, owing to the size of the containers, limit the number of possible “reaction chambers” to a few hundred that can potentially yield data. qPCR also requires users to repeat the experiment a few times using the sample diluted a known number of times to derive the mathematical curve since too much or too little of the target mutation or reagent chemicals and other substances can skew the results. In contrast, ddPCR generates tens of thousands of discrete droplets of an almost identical nanolitre size containing a mixture of the sample, reagents and stabilising chemicals. ddPCR then analyses the droplets to determine if at least 1 copy of the mutation is present or not in each droplet. Poisson statistics is then used to calculate the total number of copies of the mutation in the sample. Since each droplet is informative, the sensitivity and accuracy of ddPCR are much greater than that of qPCR.
To study whether ddPCR could indeed be a more sensitive diagnostic and monitoring tool for the IDH1 mutation, Dr To and his collaborators obtained consent from 62 mainland Chinese patients with grades II to IV of glioma to test biopsied samples from their brain tumour specimens using ddPCR, qPCR, and Sanger direct sequencing as a benchmark. Although Sanger sequencing is usually not used in hospitals and cannot quantify, it is currently the gold standard for discovering the exact sequence of bases — and therefore the locations of mutations — in genetic material.