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Restriction endonuclease‐mediated real‐time digestion‐PCR for somatic mutation detection
Author(s) -
Zhao Jinyin,
Xie Feifei,
Zhong Wei,
Wu Weili,
Qu Shoufang,
Gao Shangxian,
Liu Licheng,
Zhao Jing,
Wang Mengzhao,
Zhou Junyi,
Jie Huang,
Chen Weijun
Publication year - 2012
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.27968
Subject(s) - cold pcr , sanger sequencing , microbiology and biotechnology , biology , endonuclease , taqman , variants of pcr , restriction enzyme , somatic cell , point mutation , restriction digest , t790m , mutant , mutation , digital polymerase chain reaction , polymerase chain reaction , genetics , dna , gene , kras
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.