title: Detection of low-level DNA mutation by ARMS-blocker-Tm PCR.
abstract: BACKGROUND
  Low-level DNA mutations play important roles in cancer prognosis and treatment. However, most existing methods for the detection of low-level DNA mutations are insufficient for clinical applications because of the high background of wild-type DNA.
  
  
  METHODS
  In this study, a novel assay based on Tm-dependent inhibition of wild type template amplification was developed. The defining characteristic of this assay is an additional annealing step was introduced into the ARMS-blocker PCR. The temperature of this additional annealing step is equal to the Tm of the blocker. Due to this additional annealing step, the blocker can preferentially and specifically bind the wild-type DNA. Thus, the inhibition of wild type template is realized and the mutant DNA is enriched.
  
  
  RESULTS
  The sensitivity of this assay was between 10(-4) and 10(-5), which is approximately 5 to 10 times greater than the sensitivity of the assay without the additional annealing step. To evaluate the performance of this assay in detecting K-ras mutation, we analyzed 100 formalin-fixed paraffin-embedded (FFPE) specimens from colorectal cancer patients using this new assay and Sanger sequencing. Of the clinical samples, 27 samples were positive for K-ras mutation by both methods.
  
  
  CONCLUSIONS
  Our results indicated that this new assay is a highly selective, convenient, and economical method for detecting rare mutations in the presence of higher concentrations of wild-type DNA.
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: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Dongdajie Road 20, Beijing 100071, China.
authors.forename: Licheng
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beitucheng West Road 11, Beijing 100029, China.
authors.forename: Shuzhen
authors.initials: S
authors.lastname: Gan
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beitucheng West Road 11, Beijing 100029, China.
authors.forename: Huahua
authors.initials: H
authors.lastname: Feng
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beitucheng West Road 11, Beijing 100029, China.
authors.forename: Jingyin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Respiratory Medicine, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Beijing Macro & Micro Test Biotech Company, Beijing 101312, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Division of in Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China.
authors.forename: Shangxiang
authors.initials: S
authors.lastname: Gao
authors.affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beitucheng West Road 11, Beijing 100029, China.
authors.forename: Weijun
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Respiratory Medicine, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China. Electronic address: mengzhaowang@sina.com.
authors.forename: Mengzhao
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Dongdajie Road 20, Beijing 100071, China. Electronic address: jiangyq710327@163.com.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Division of in Vitro Diagnostic Reagents, National Institutes for Food and Drug Control (NIFDC), Beijing 100050, China. Electronic address: jhuang5522@126.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D009154:Mutation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D018631:ras Proteins / Q000235:genetics
chemical_list: D004247:DNA; D018631:ras Proteins
keywords: ARMS; Blocker; Enrich; Rare mutations