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 1. Rare allele measurement using spike-recovery method. Differing amounts of DNA from three different oncogenic KRAS alleles were spiked into a constant amount of normal DNA. Note the excellent correlation between input concentration and measured concentration; the linear slope indicates that the amounts of mutant allele were accurately measured.
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.

Digital PCR: Rare Mutation Applications on the QuantStudio™ 3D

Genetic mutation is a hallmark of cancer, and the ability to detect genomic DNA changes is paramount to understanding this disease at a molecular level. Assays developed to detect rare mutations need to be able to identify just a few mutant cells in a huge abundance of wild type cells. Digital PCR combined with TaqMan SNP Genotyping Assays offer a highly sensitive and specific solution for the confirmation of these discoveries.