title: A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis.
abstract: BACKGROUND
  We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer.
  
  
  METHODS
  DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells.
  
  
  RESULTS
  The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%.
  
  
  CONCLUSIONS
  PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.
authors.affiliation: Second Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan. tnishika@mdanderson.org
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishikawa
authors.forename: Kentaro
authors.initials: K
authors.lastname: Maemura
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirata
authors.forename: Ryouichi
authors.initials: R
authors.lastname: Matsuse
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Morikawa
authors.forename: Ken
authors.initials: K
authors.lastname: Toshina
authors.forename: Mitsuyuki
authors.initials: M
authors.lastname: Murano
authors.forename: Keiichi
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Nakagawa
authors.forename: Osamu
authors.initials: O
authors.lastname: Saitoh
authors.forename: Kazuo
authors.initials: K
authors.lastname: Uchida
authors.forename: Kenichi
authors.initials: K
authors.lastname: Katsu
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D005243:Feces / Q000737:chemistry*; D005260:Female; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D017354:Point Mutation / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D004247:DNA