DENATURING GRADIENT GEL-ELECTROPHORESIS (DGGE) ASSAY FOR K-RAS AND N-RAS GENES - DETECTION OF K-RAS POINT MUTATIONS IN HUMAN LUNG-TUMOR DNA

Point mutations in the ras oncogenes are very common in lung cancers as well as in many of the other solid tumours. To effectively examine the occurrence of these mutations in a large number of tumour samples, we have applied denaturing gradient gel electrophoresis (DGGE) for the analysis of point mutations of the K-ras and N-ras genes, using GC-clamped, PCR-amplified DNA fragments. Among the 68 tumour DNA samples, we detected 14 mutations in the K-ras gene. This was 78% of the mutations identified by oligonucleotide hybridization. Altogether, eight of the nine different kinds of base substitutions found in the tumour samples were detected by the DGGE assay, representing substitutions at codons 12, 13, and 61 of the K-ras gene. Six of the detected mutations were guanine to thymine transversions at codon 12; this was the most common type of alteration. On the basis of our experience, the present non-radioactive DGGE analysis seems to be readily applicable for detection of the mutations in the K-ras and N-ras genes. Types of ras gene mutations frequent in adenocarcinomas of the lung are also discussed.