A fast, cost-efficient and sensitive approach for KRAS mutation detection using multiplexed primer extension with IP/RP-HPLC separation

Mutations in the KRAS gene are very important diagnostic and prognostic markers in cancer. Particularly, KRAS mutations at codons 12 and 13 have a high prognostic value for EGFR-directed antibody therapies. Several methods are available to detect the most common mutations, some of them are commercialized. The most frequently used techniques, allele-specific PCR or direct sequencing, are not standardized and often lack sensitivity to detect low amounts of mutated tumor cells in paraffin-embedded tissue-blocks leading to a high number of false-negatives. Here we present a reliable, fast, cost-effective and sensitive approach for KRAS mutation detection that has a high potential for standardized large scale screening. The method is based on multiplexed primer extension reactions coupled to HPLC separation. The highly sensitive assay gives easily interpretable and reproducible results at affordable costs. We describe the method and an application example for diagnosis in early colorectal cancer screening.