An alteration in copy number state with respect to a single-copy reference locus is referred to as a “copy number variation” (CNV) if it appears in germline cells, or a copy number alteration (CNA) if it appears in somatic cells. A CNV or CNA could be due to a deletion or amplification of a locus with respect to the number of copies of the reference locus present in the cell, and together, they are major contributors to variability in the human genome. They have been associated with cancers; neurological, psychiatric, and autoimmune diseases; and adverse drug reactions. However, it is difficult to measure these allelic variations with high precision using other methods such as qPCR, thus making phenotypic and disease associations with altered CNV status challenging.
The large number of “digitized,” endpoint measurements made possible by sample partitioning enables dPCR to resolve small differences in copy number with better accuracy and precision when compared to other methods such as SNP-based microarrays or qPCR. qPCR is limited in its ability to precisely quantify gene amplifications in several diseases, including Crohn’s disease, HIV-1 infection, and obesity.
dPCR was designed to measure the concentration of a nucleic acid target in copies per unit volume of the sample. When operating in dilute reactions where less than ~10% of the partitions contain a desired target (referred to as “limiting dilution”), copy number can be estimated by comparing the number of fluorescent droplets arising from a target CNV with the number of fluorescent droplets arising from an invariant single-copy reference locus. In fact, both at these lower target concentrations and at higher ones where multiple copies of the same target can co-localize to a single partition, Poisson statistics are used to correct for these multiple occupancies to give a more accurate value for each target’s concentration.
Digital PCR has been used to uncover both germline and somatic variation in gene copy number between humans and to study the link between amplification of HER2 (ERBB2) and breast cancer progression.