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

Abstract

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. What's new? The polymerase chain reaction (PCR) enables the detection of DNA mutations in large pools of wild-type sequences. However, while PCR-based methods have advanced significantly, they remain relatively insensitive to minority alleles and low-level mutations. Copyright © 2012 UICC.