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

Clin Chim Acta. 2002 Apr;318(1-2):107-12. doi: 10.1016/s0009-8981(01)00806-3.

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.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Colorectal Neoplasms / diagnosis*
  • Colorectal Neoplasms / genetics*
  • DNA / genetics
  • DNA / isolation & purification
  • DNA Primers
  • Feces / chemistry*
  • Female
  • Genes, ras / genetics*
  • Humans
  • Male
  • Middle Aged
  • Point Mutation / genetics*
  • Polymorphism, Restriction Fragment Length
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tumor Cells, Cultured

Substances

  • DNA Primers
  • DNA