Common SNP genotyping technologies, such as capillary electrophoresis (CE) sequencing and real-time PCR, are most effective at detecting mutant cells with a prevalence no lower than about 20% (or approximately 1 in 5 cells). By combining real-time PCR chemistries, such as TaqMan® Assays, with digital PCR methodology, researchers are now able to detect mutant cell prevalence down to 1%—and below.
Digital PCR works by partitioning a sample into many individual reactions prior to amplification, reducing competing wild type sequences in any reaction containing a mutation and effectively decreasing background noise. If sufficient partitions are used, the reaction wells reach a point where the wild-type signal no longer overwhelms the mutant signal. Because each data point is generated digitally, the total count of each allele, mutant and wild type, can be calculated and a ratio determined.
Figure 2. Allelic chimerism in bone marrow transplant samples. Two alternate alleles that differentiated a bone marrow donor from a recipient were chosen. Samples were collected pre-stem cell transplant (pre-SCT) and at the indicated times after transplant. Note the recipient's allele starts to reappear after 101 days, and is obvious by 118 days, indicating a relapse.