Mutation analyses of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillaryelectrophoresis and allele specific polymerase chain reaction

Abstract

Mutations in the KRAS gene is a key event in the carcinogenesis of many human cancers and may serve as a diagnostic marker and a target for therapeutic intervention. In this study we have applied three different techniques for mutation detection of KRAS exon 1 mutations: Allele specific polymerase chain reaction (AS-PCR), temporal temperature gradient electrophoresis (TTGE) and constant denaturant capillary electrophoresis (CDCE). Samples from 191 sporadic colon carcinomas were analyzed. AS-PCR were performed with oligonucleotides specific for know mutations in codon 12 and 13 of the KRAS gene. In TTGE analyses, linear ramping of the temperature were performed during electrophoresis in a constant denaturant gel. CDCE analyses were performed using fluorescin labeled PCR-products. Separation was achieved under constant denaturing conditions using high temperature in a gel-filled capillary followed by laser detection. A mutated KRAS gene was found in 42 191 (22.0%) of the samples using AS-PCR, in 62 191 (32.5%) using TTGE and in 66 191 (34.6%) of the samples using CDCE. In the TTGE and CDCE analyses the sequence of the mutant were determined by comparing the electrophoretic pattern to that of known mutations or by mixing the sample with known mutations prior to reanalysis. In a titration experiment mixing mutant and wild-type alleles prior to PCR, the sensitivity for mutation detection was shown to be 10 −2 for TTGE and under optimized conditions 10 −3 for CDCE.