Sensitive mutation detection for cancer research
Rare mutation detection has great implications in areas such as cancer research because the accumulation of mutations in crucial regulatory genes, such as oncogenes or tumor suppressor genes, is an important aspect of tumorigenesis. Acquisition of these mutations in a tiny subset of somatic cells can be sufficient for cancer initiation or progression.
Since these mutations are so rare, they require an assay that delivers high signal-to-noise and low false-positive-to-false-negative rates.
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.
For Research Use Only. Not for use in diagnostic procedures.