Rapid Detection of H-RAS Point Mutation Using Two-Step Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

  • Park, Young-Suk (Department of Molecular Genetics, Seoul Medical Science Institute (SeL)) ;
  • Lee, Kyung-Ok (Department of Molecular Genetics, Seoul Medical Science Institute (SeL)) ;
  • Chai, Young-Gyu (Department of Biochemistry, Hanyang University)
  • Received : 1996.05.15
  • Published : 1996.09.30

Abstract

Mutations in codon 12, 13 and 61 of one of the three ras genes, H-ras, K-ras and N-ras, convert these genes into active oncogenes. The presence of H-ras gene mutations have important prognostic implications in various cancers. In this study, the H-ras gene mutations were investigated by two-step PCRRFLP in patients with bladder and stomach cancer. For the control experiments, T24 and SK2 cell lines were used. In a total of 36 bladder cancer patient cases, five (13.9%) mutations were found by this method. Of these, point 12 mutations were two (5.6%) cases and point 61 mutations were three (8.3%) cases. On the other hand, H-ras mutation was not found in 29 cases of stomach cancer. The results of the mutated H-ras gene confirmed by direct sequencing analysis were correlated well with PCR analysis. From the sensitivity test, the H-ras mutation was found to have about 0.2% of mutated DNA mingled in normal DNA. In conclusion, the H-ras mutation has a higher clinical Significance in bladder cancer than stomach cancer. Moreover the two-step PCR-RFLP method is sensitive, rapid and relatively simple for clinical work in detecting H-ras point mutations.

Keywords

H-ras point mutation;two-step PCR-RFLP

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