New comprehensive denaturing-gradient-gel- electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours.

A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within two lanes of a DGGE gel under a single set of experimental conditions. Six known codon 12 mutations in genomic DNA from different paraffin-embedded tumours could readily be detected. When testing 35 paraffin-embedded Dukes' C colorectal carcinomas for unknown mutations, 12 tumours were found with mutations in codons 12 or 13. None of the tumours appeared to have a codon 61 mutation. In nine tumours, however, 11 additional sequence variations were found outside the hot-spot codons. None of these occurred in germline DNA from 57 individuals. Of these variations, three are considered as significant mutations, as they result in a non-conservative substitution of amino acid residues essential for the functioning of the KRAS protein. Thus, in total, 15 of the 35 Dukes' C tumours (43%) had a KRAS mutation of functional significance. Moreover, a novel exon 4B polymorphism was found to occur in 10 of the 35 tumours. The results of this study suggest that in restricting analysis of KRAS to hot-spot mutation sites only, significant information may be missed. Copyright 2000 Wiley-Liss, Inc.