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 ba sed on denaturing gradient gel electrophoresis and applicable to archival p araffin-embedded tumour material. All KRAS amplicons are analysed within tw o lanes of a DGGE gel under a single set of experimental conditions. Six kn own codon 12 mutations in genomic DNA from different paraffin-embedded tumo urs could readily be detected. When testing 35 paraffin-embedded Dukes' C c olorectal carcinomas for unknown mutations, 12 tumours were found with muta tions in codons 12 or 13. None of the tumours appeared to have a codon 61 m utation. In nine tumours, however, I I additional sequence variations were found outside the hot-spot codons. None of these occurred in germline DNA f rom 57 individuals. Of these variations, three are considered as significan t mutations, as they result in a non-conservative substitution of amino aci d residues essential for the functioning of the KRAS protein. Thus, in tota l, 15 of the 35 Dukes' C tumours (43%) had a KRAS mutation of functional si gnificance. 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 a nalysis of KRAS to hot-spot mutation sites only, significant information ma y be missed. (C) 2000 Wiley-Liss, Inc.