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

Takashi Nishikawa, Kentaro Maemura, Ichiro Hirata, Ryouichi Matsuse, Hiroshi Morikawa, Ken Toshina, Mitsuyuki Murano, Keiichi Hashimoto, Yoshihito Nakagawa, Osamu Saitoh, Kazuo Uchida, Kenichi Katsu

Research output: Contribution to journalArticle

38 Citations (Scopus)

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 ≥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.

Original languageEnglish
Pages (from-to)107-112
Number of pages6
JournalClinica Chimica Acta
Volume318
Issue number1-2
DOIs
Publication statusPublished - 14-03-2002
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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