Rare Event Detection
Rare events include single nucleotide mutation, alteration of copy number, and deletion or insertion of nucleotides. These genetic variant molecules are difficult to detect due to their dilution by normal cells from either tissues or bodily fluids such as blood. Early detection of rare events can make all the difference in the outcome of cancer patients, as well as lead to more sensitive and less invasive diagnostics.
During qPCR, rare and normal variants of DNA molecules are amplified at an equivalent rate. If the normal gene is initially present in 100-fold abundance over the mutated gene, at the end of the reaction, 1% of the total amplification product will be the mutated gene amplicon. Its detection will be extremely difficult as the total signal generated will be 1% of that generated by the wild-type gene amplicon.
For rare event detection, sample partitioning increases sensitivity by distributing both mutant and normal genes into a large number of isolated reaction compartments. In each one of these isolated droplets, the rare mutant molecules are now at a more favorable ratio compared to the wild-type, and thus become detectable within that individual droplet. The QX100 can detect amplifications even in highly heterogeneous matrices where only a fraction of the cells are affected. This precision enables the detection of somatic copy number alteration—the hallmark of many cancers.
The detection of point mutations requires a high degree of sensitivity. Droplet Digital PCR allows the detection of 0.001% mutation fractions, as demonstrated in a duplex PCR reaction using TaqMan probes targeting the BRAF 600E mutation (Figure 2). This detection limit is more than 1,000 times lower than qPCR.