Genetic Analysis of Paraffin-Embedded Human Tumor Samples by GC-clamped DGGE.

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We established the GC-clamped DGGE method which can detect genetic alterations in small pieces of tissue samples without use of radioisotopes. This method was applicable to analysis of human tumors enucleated from tissue sections. In practice, GC-rich sequences were introduced to one side of PCR fragments using GC-clamped PCR primers. When these PCR products were electrophoresis on gels which vertically contained a 0-80% gradient of denaturing agents (urea and formamide), electrophoretic mobility was retarded over a certain concentration of the denaturants at which conformational changes of DNA occurred. When the PCR products contained mutated sequences, electrophoresis patterns were differed to that of the wild type cases. Many samples can be simultaneously analyzed by electrophoresing the samples to the parallele direction of gradient.It was revealed that the method was sensitive enough to detect mutations in one part of mutated DNA diluted with 16 parts of wild type DNA.Following results were obtained using this method.1)K-ras codon 12 mutations were highly frequent in human bile duct and gallbladder carcinomas. These mutations were mainly a glycine to serine change.2)K-ras and p53 mutations were compared between mucin-producing cystic type and ordinary form of human pancreatic cancers. Although K-ras codon 12 mutations were detected in most cases of both types, p53 mutations were found only in the ordinary form.3)p53 mutations were highly frequent in rat hepatocellular carcinoma cell (HCC) lines. However, such mutations were rarely observed in chemically-induced rat HCCs, indicating that the p53 mutations may occur during establishment of HCC cell lines.4)K-ras condon 12 mutations were detected at low frequency in rat HCCs. However, frequency of the mutations was different depending on HCC-inducing agents.5)p53 mutations were rarely detected in spontaneously-immortalized mouse embryonic fibroblasts and their transformed derivatives.