Restriction endonuclease‐mediated real‐time digestion‐PCR for somatic mutation detection

PCR is a powerful platform for clinical and diagnostic applications, but challenges remain in detecting somatic mutations, as mutant cells are often mixed with more numerous wild-type cells at the tissue-sample sites. Here, we describe a novel method that couples PCR with restriction endonuclease digestion (designated real-time digestion-PCR, or RTD-PCR) in a one-step reaction tube for detecting somatic mutations from a minority of cells. The PCR mixture contains a thermostable restriction enzyme that digests wild-type alleles during the PCR program, allowing selective amplification of the mutant alleles. To validate this method, we used real-time digestion-PCR for the specific detection of the EGFR (epidermal growth factor receptor) treatment resistance-inducing mutation, T790M, combining with three different platforms: Sanger sequencing, TaqMan probe PCR and Sequenom MassArray. From 78 clinical samples, seven T790M mutations were consistently detected on all three platforms, indicating that RTD-PCR may be a useful clinical tool for analyzing the T790M point mutation.