Mutation Detection in KRAS Exon 1 by Constant Denaturant Capillary Electrophoresis in 96 Parallel Capillaries

Abstract

Mutations in KRAS exon 1 oncogene are frequently found in colon carcinomas. A correlation between the mutated KRAS and the prognosis and outcome of treatment of colon cancer patients was reported in the literature. The object of our work was to establish a high-throughput method with high sensitivity to enable screening of tumor mutation status of KRAS exon 1 in large groups of colon cancer patients. KRAS exon 1 sequences from DNA isolated from 191 sporadic colon cancers were PCR amplified using one primer labeled with fluorescein and a second primer extended by a GC-clamp. After PCR amplification samples were subjected to automated 96-array constant denaturant capillary electrophoresis using a modified MegaBACE 1000 sequencing instrument. Mutant samples were identified by characteristic peak patterns. The sensitivity of detection of a mutant allele in a background of the wild-type alleles was 0.3%. Using the 96-array instrument a typical screening of 191 samples for KRAS mutation status could be performed within 2 h. A KRAS exon 1 mutation was found in 66 of 191 (34.6%) of the samples. The 96-array constant denaturant capillary electrophoresis provides an opportunity for the high-sensitivity screening of large cancer populations for KRAS exon 1 mutations.