Abstract 2116: High-sensitivity K-RAS mutation detection using a plant endonuclease-based genotyping method

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Newly developed therapeutic agents targeting the epidermal growth factor receptor (EGFR) have proven to be effective against colorectal cancer. However, in certain colorectal cancers these drugs are ineffective. Around 40% of colorectal tumors carry K-RAS gene mutations associated with poor response to EGFR antagonists. K-RAS mutation status can therefore be used to determine whether or not a tumor will respond to anti-EGFR therapy. SURVEYOR Nuclease is a mismatch-specific plant DNA endonuclease that can scan for known and unknown mutations and polymorphisms in heteroduplex DNA by cleaving DNA with high specificity at sites of base-substitution mismatch and other alterations. This DNA endonuclease cuts both strands of a DNA heteroduplex on the 3’-side of the mismatch site, yielding fragments of smaller size that can be detected by size discrimination technologies. Small insertions/deletions as well as all base-substitution mismatches are recognized; the efficiency of cleavage varies with the sequence of the mismatch. SURVEYOR Scan K-RAS is a research-use-only kit for detecting all sequence variations in exons 2 and 3 of the K-RAS gene. Studies using plasmid controls have demonstrated detection of all known activating exon 2 mutations in codons 12 and 13, as well as the most common mutation in exon 3, Q61H. Limit of detection experiments have demonstrated that SURVEYOR Scan is highly sensitive, capable of detecting a mixture of 1% mutant in a background of 99% non-mutant DNA. The resulting digestion patterns for codons 12, 13 and 61 are highly specific and recognizable by comparison with controls. In addition, since heteroduplex cleavage is not position specific, mutations outside of these three codons also are detected. SURVEYOR Scan has been used successfully with HPLC (WAVE® HS System) and with automated micro-fluidic capillary electrophoresis (Caliper Lab Chip GX) suitable for high-throughput analyses. Use of SURVEYOR Scan K-RAS can decrease sequencing burden and aid aggressive sequence calling where automated sequencing software fails to definitively resolve the presence of a mutation. This method has also been successfully applied to activating mutations in the BRAF and PIK3CA genes as well. The extension of SURVEYOR Scan to mutation analysis in tumor suppressor genes, such as p53, in which inactivating mutations may be present in multiple exons in numerous locations, is anticipated to be of significant utility, since mutation-specific methods are not practical and sequencing requirements are onerous. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2116.