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Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFB1)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with allele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G.C-to-A.T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G.C-to-T.A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G.C-to-T.A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepato-cellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.

proceedings of the national academy of sciences of the united states of america

Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.