Background

The trend of molecular diagnostics is continuing to grow and allows to identify diseases at a genetic level. Measuring levels of specific gene mutations can provide a specific therapy for an individual patient by stratifying the disease status and selecting the proper medication and tailoring dosages. This personalized medicine reduces the individual risk for adverse side effects and also helps to reduce the pressure on health care costs. Successful personalized treatments are practiced in the field of oncology. The KRAS and the BRAF gene are important diagnostic and prognostic markers in colon cancer. A mutation screening of these genes has to be performed prior to the selection of patients which are candidates for EGFR-I treatment. The two anti-EGFR monoclonal antibody drugs (panitumumab (Vectibix) and cetuximab (Erbitux)) are only approved in the European Union for the treatment of patients with EGFR-expressing metastatic cancer of the KRAS wild-type.

Invention and Application

Scientists of the Saarland University and the Charité Berlin established an assay which allows the selective detection of all 14 clinically relevant variants of the KRAS gene (codon 12 and 13) and the BRAF gene (codon 600-2). Amplicons obtained from a multiplexed PCR are used for multiplexed primer extension reactions, followed by a multiplexed HPLC separation. Compared to the most frequently used technique of direct sequencing, the assay shows a much higher sensitivity with a reliable detection limit of 5%, compared to 20% of mutated DNA in a wild type background, with causing only one third of the costs of direct sequencing. Compared to other methods like pyrosequencing, real-time PCR, ARMS/S or LCN/HRM, the assay does not require complicated establishment procedures, expensive lab automation or technical replicates.

Advantages

• simple, cost effective, fast
• semi-automatable procedure, genotyping of 50 individual DNA´s in 24 h
• robust and reliable method, no replicates needed
• no labelling or complex chemistry required
• transferable to other oncogenes; possible assay combination of different genes in one reaction