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With the new LifeTechnologies developed castPCR technology, somatic mutations now have nowhere to hide. The TaqMan® Mutation Detection Assays use this castPCR™ technology, (which refers to competitive allele-specific TaqMan® PCR) are specifically designed to detect somatic mutations that occur in cancer cells.
Let's see how it works. Let's take an example of a tumor sample in which only 0.1% of the DNA contains a somatic mutation and the other 99.9% is normal, wild type DNA . A mutant allele assay based on castPCR technology contains four components: The allele specific (AS) primer is designed to be specific for a mutant allele in a target gene. A locus specific primer is designed to be specific to the target gene. This will bind to mutant and wild type allele-containing DNA. A locus specific TaqMan® probe is designed to bind to the target gene between the primers. This will also bind to both mutant and wild type allele-containing DNA. This probe will generate a fluorescent signal during the PCR reaction to monitor the target amplification. An allele specific MGB blocker oligonucleotide is designed to bind specifically to the wild type allele. The role of this blocker is to compete with the mutant allele specific primer and inhibit its binding to the wild type allele. This blocker suppresses non specific amplification of the wild type allele and improves the specificity of the reaction.
Only the mutant allele is amplified and generates a florescent signal. By increasing the specificity and inhibiting the amplification of the wild type allele, this competitive allele-specific TaqMan® PCR provides a very sensitive and specific method to detect as low as 1 mutant allele in a presence of 1000 wild type alleles.