title: Sensitive detection of trace amounts of KRAS codon 12 mutations by a fast and novel one-step technique.
abstract: OBJECTIVE
  The objective of this study is to develop a novel and sensitive method for KRAS codon 12 mutation testing.
  
  
  METHODS
  We developed a sensitive one-step real-time digestion-and-block TaqMan probe PCR (RTDB-PCR) technique that uses a thermostable endonuclease and a minor groove binder (MGB) blocker to detect KRAS codon 12 mutations. Dilution mimic DNA panels were used to assess the sensitivity of this technique. The RTDB-PCR method was performed and compared with three other methods: PCR sequencing, mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray. A total of 100 formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) specimens were also tested by all four methods.
  
  
  RESULTS
  The RTDB-PCR was sensitive to as little as 0.01% mutant DNA, significantly higher than other methods. Among the 100 FFPE mCRC specimens examined, 45 tested positive for KRAS codon 12 mutations according to RTDB-PCR, 44 tested positive according to mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, and only 26 samples tested positive according to PCR sequencing.
  
  
  CONCLUSIONS
  Compared with mutant-enriched PCR sequencing and mutant-enriched PCR-MassArray, RTDB-PCR is more cost effective, saves time, and is easier to use, making it suitable for the detection of low-level KRAS mutations in the clinic.
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Xie
authors.affiliation: Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
authors.affiliation: Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China.
authors.forename: Shoufang
authors.initials: S
authors.lastname: Qu
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Weili
authors.initials: W
authors.lastname: Wu
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Huagui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Wang
authors.affiliation: Beijing Macro & Micro Test Biotech Company, Beijing 101312, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Beijing Macro & Micro Test Biotech Company, Beijing 101312, China.
authors.forename: Senlin
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Beijing Macro & Micro Test Biotech Company, Beijing 101312, China.
authors.forename: Lizhi
authors.initials: L
authors.lastname: Xu
authors.affiliation: Division of In Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China.
authors.forename: Shangxian
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Dermatology, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou, Guang Dong Province 510630, China.
authors.forename: Yunqing
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: zhaojy526@gmail.com.
authors.forename: Jinyin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: chenwj@genomics.org.cn.
authors.forename: Weijun
authors.initials: W
authors.lastname: Chen
mesh_terms: D003062:Codon / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D009154:Mutation; D015203:Reproducibility of Results; D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; D018631:ras Proteins
keywords: Colorectal cancer; KRAS mutation detection; Low-level mutation