Somatic mutations are the most common cause of cancer. These mutations occur in any of the cells of the body except the germ cells, therefore, are not passed on to the next generation. Cancers that occur because of somatic mutations are referred to as sporadic cancers.
TaqMan™ Mutation Detection Assays are powered by competitive allele-specific TaqMan™ PCR (castPCR™ Technology) to detect and measure somatic mutations in genes associated with cancer research. The castPCR technology is highly specific and sensitive, and can detect rare amounts of mutated DNA in a sample that contains large amounts of normal, wild-type DNA.
By combining the power of TaqMan and castPCR technologies, TaqMan Mutation Detection Assays give you:
- Higher specificity – designed to block the wild-type and amplify only the mutant
- Higher sensitivity – designed to detect somatic mutations down to 1 mutant allele in the presence of 1000 wild type alleles.
- Faster workflow – 3 hours from sample to result
With 819 assays for 47 known cancer research genes available today like KRAS, BRAF, KIT, JAK2, the TaqMan Mutation Detection Assays provide an innovative tool for your cancer research.
Somatic Mutation Detection Workflow:
1. Sample Type Compatibility
The assays can be used with gDNA extracted from FFPE tissues, fresh frozen tissues, and cell line samples.
2. Mutant Allele Assays
Mutant allele assays target key somatic mutations in oncogenes and tumor suppressor genes. All mutation targets are from the comprehensive Sanger COSMIC database (www.sanger.ac.uk/genetics/CGP/cosmic/). Target selection was based on frequency of occurrence and input from leading cancer researchers. Each assay contains:
- a locus-specific pair of forward and reverse primers
- a locus specific TaqMan® FAM™ dye-labeled MGB probe
For each real-time PCR reaction, the gDNA is combined with:
- A TaqMan® Mutation Detection Assay—to detect a mutant allele or reference gene target
- TaqMan® Genotyping Master Mix
- Optional Use of Internal Positive Control (IPC)—add IPC reagents to any mutant allele assay reaction to distinguish true target negatives from PCR failure or inhibition.
Reactions are run on a real-time PCR system using a universal mutation detection termal cycling protocol.
Data files containing the sample Ct values can be exported from instrument software and imported into Mutation Detector™ Software for post-PCR data analysis of mutation detection experiments. In mutation analysis calculations, the difference between the Ct for each mutant allele assay and the Ct for the gene reference assay is calculated. This ΔCt value represents the quantity of the specific mutant allele detected within the sample.
The power of digital PCR can also be used to detect rare mutations for cancer research. Using the QuantStudio™ 3D Digital PCR System and wet-lab validated TaqMan SNP Genotyping Assays, you can target over 100 somatic mutations at a prevalence as low as 0.1%.