Detection of H-ras gene point mutations in transitional cell carcinoma of human urinary bladder using polymerase chain reaction.

Abstract

It has been reported that the H-ras gene is activated as oncogene in human bladder cancer cases, and that codon 12 and codon 61 are the major "hot spots" of its activation. A simple method to detect point mutations in these codons of H-ras gene was established for the use of clinical diagnosis. In this method, the DNA segments including codon 12 or codon 61 were amplified by polymerase chain reaction (PCR), and the reaction products were examined for their susceptibility to the restriction enzyme NaeI or BstNI, and by dot blot hybridization assay with oligonucleotide probes. Point mutations were detectable in small amounts of DNAs isolated from fresh or frozen tumor tissues, urine cells and paraffin sections. The method was applied for a clinical sample and a case that had a point mutation at codon 12 of H-ras gene was detected. The point mutation was existed in DNAs of primary tumor tissue, all recurrent tumor tissues and cells isolated from urine of this case.