title: 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.
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Jinyin
authors.initials: J
authors.lastname: Zhao
authors.forename: Feifei
authors.initials: F
authors.lastname: Xie
authors.forename: Wei
authors.initials: W
authors.lastname: Zhong
authors.forename: Weili
authors.initials: W
authors.lastname: Wu
authors.forename: Shoufang
authors.initials: S
authors.lastname: Qu
authors.forename: Shangxian
authors.initials: S
authors.lastname: Gao
authors.forename: Licheng
authors.initials: L
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Mengzhao
authors.initials: M
authors.lastname: Wang
authors.forename: Junyi
authors.initials: J
authors.lastname: Zhou
authors.forename: Huang
authors.initials: H
authors.lastname: Jie
authors.forename: Weijun
authors.initials: W
authors.lastname: Chen
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D004252:DNA Mutational Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes; D066246:ErbB Receptors / Q000235:genetics; D005091:Exons; D005838:Genotype; D006801:Humans; D009154:Mutation
chemical_list: D066246:ErbB Receptors; D004262:DNA Restriction Enzymes