Point Mutation of the ras Oncogene in Human Ovarian Cancer

Abstract

The ras oncogene exists in a variety of human cancers, including carcinomas of bladder, breast, colon, kidney, liver, lung, ovary, pancreas, and stomach. The ras genes acquire transforming activity either by enhanced expression or by a single point mutation. A single base-pair mutation at specific sites within ras genes endows them with the capacity to transform certain cell lines in vitro. In this study, we showed the patterns of point mutations in codons 12, 13, and 61 of ras genes in human ovarian cancer. The experimental procedures were isolation of genomic DNA from normal ovary and ovarian cancer tissue specimens, amplification of a genomic DNA segment (about 100 bp) using different 5′ and 3′ extension primers in the polymerase chain reaction (PCR), labeling and purification of synthetic mutation-specific oligonucleotide probes, slotblot hybridization, and autoradiography. The three reaction steps for the PCR cycle were: 96°C for 1 min in step 1, 56°C for 1 min in step 2, and 74°C for 1 min in step 3. The PCR reaction was repeated totally for 30 cycles. In 28 tissue specimens of human ovarian cancer examined, one specimen was found with a c-Ha-ras point mutation at codon 12, two had a c-Ki-ras mutation at codon 12, and one had a c-Ki-ras mutation at codon 13.