Detecting Rare Cancer Mutations With castPCR™ Technology

TaqMan® Mutation Detection Assays

In cancer tissue samples, somatic mutations are often present in vanishingly small amounts when compared with the high levels of wild-type DNA sequences. Detecting these rare mutations in the presence of such a high background of wild-type alleles can be challenging. Existing mutation detection methods compatible with tumor specimens are often limited by poor sensitivity and specificity, high cost, and inconvenient workflows. TaqMan® Mutation Detection Assays were designed to address these limitations, as well as to provide a method for accurately quantifying the percentage of specific cancer mutations within a sample.

Competitive PCR Assays With castPCR™ Technology

Our TaqMan® Mutation Detection Assay portfolio covers key somatic mutations identified in various cancer genes—including BRAF, EGFR, HRAS, KIT, KRAS, NRAS, PIK3CA, PTEN, and TP53—and implicated in many types of cancer. TaqMan® Mutation Detection Assays take advantage of our pioneering Competitive Allele–Specific TaqMan® (castPCR™) technology, which combines mutant allele–specific TaqMan® qPCR amplification assays with wild-type allele–specific MGB blocker oligonucleotides that effectively suppress nonspecific amplification of the wild-type allele (for more details, see our new video at lifetechnologies.com/castpcr). By combining mutant allele–specific primers with the proprietary MGB blocker oligonucleotides, these assays show a high degree of specificity, enabling the detection of as little as 0.1% mutant allele in the presence of a wild-type allele background (Figure 1).

TaqMan® Mutation Detection Assays are not only sensitive but also efficient, with a wide dynamic detection range that spans over four logs in template concentration. Moreover, the TaqMan® Mutation Detection Assays produce reproducible and accurate quantification, with an average amplification efficiency of 100% ± 10%. TaqMan® Mutation Detection Assays are compatible with genomic DNA samples extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, fresh-frozen tissue, and cell culture.

Figure 1. Detection of KRAS 518 mutant alleles using the TaqMan® Mutation Detection Assay. The amplification plot shows the Ct difference between a 0.1% mutation sample (10 copies mutant allele and 30 ng wild-type gDNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue) and a wild-type gDNA sample (30 ng wild-type gDNA isolated from FFPE tissue). Both samples were amplified using the TaqMan® Mutation Detection Assay for the KRAS 518 mutation. The 0.1% mutation sample was created by adding gDNA from the mutant cell line PSN-1, which carries the KRAS 518 mutation, to gDNA from the wild-type Jurkat cell line.

State-of-the-Art Mutation Detection

We are continually adding mutation assays to cover newly identified cancer gene mutations. For the most updated list of available assays, refer to the TaqMan® Mutation Detection Assay index, which currently contains 586 different mutations in 45 different genes.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.