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.

 

Detection of KRAS 518 mutant alleles using the TaqMan Mutation Detection Assay.  
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.

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