title: Detection of KRAS codon 12 and 13 mutations by mutant-enriched PCR assay.
abstract: BACKGROUND
  The identification of KRAS mutations before the administration of anti-epidermal growth factor receptor (EGFR) therapy of metastatic colorectal cancer (mCRC) has become important. The aim of the present study was to develop a novel technology that can increase detection sensitivity for KRAS mutations.
  
  
  METHODS
  DNAs were extracted from colorectal cancer tissues and formalin-fixed, paraffin-embedded (FFPE) colorectal cancer samples. Mutant-enriched PCR assay utilizes the exceptionally thermostable endonucleases, PspGI for codon 12 and PhoI for codon 13, for specific amplifying KRAS mutations from mixed samples. The amplified PCR products were subjected to single-base primer extension or sequencing. Digital PCR was used to evaluate some of the results.
  
  
  RESULTS
  We compared the results with that from direct sequencing. In the FFPE samples, thirteen discordant samples were found. We showed that the mutant-enriched PCR assay can identify the codons 12 and 13 mutation in a mixed population of mutant and wild type DNA sequences at 1:1000 and 1:400, respectively. The sensitivity of this method is lower than the digital PCR.
  
  
  CONCLUSIONS
  We developed a rapid and highly sensitive method to detect codons 12 and 13 mutations of the KRAS gene. This method is a powerful tool for finding low-abundance variations in genomic DNA.
authors.affiliation: Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan; Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan.
authors.forename: Ya-Sian
authors.initials: YS
authors.lastname: Chang
authors.affiliation: Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
authors.forename: Tze-Kiong
authors.initials: TK
authors.lastname: Er
authors.affiliation: Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan.
authors.forename: Hsiu-Chin
authors.initials: HC
authors.lastname: Lu
authors.affiliation: Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan.
authors.forename: Kun-Tu
authors.initials: KT
authors.lastname: Yeh
authors.affiliation: Epigenome Research Center, China Medical University Hospital, Taichung, Taiwan; Department of Laboratory Medicine, China Medical University Hospital, Taichung, Taiwan; College of Medicine, China Medical University, Taichung, Taiwan. Electronic address: d6781@mail.cmuh.org.tw.
authors.forename: Jan-Gowth
authors.initials: JG
authors.lastname: Chang
mesh_terms: D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D015925:Cryopreservation; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: Colorectal cancer; Digital PCR; Direct sequencing; KRAS gene; Mutant-enriched PCR assay; Single-base primer extension