Background KRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer. reactions multiple actions or Erg opening PCR tubes. Methods We developed a highly sensitive single-reaction closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler? instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc) of 81°C increased the sensitivity of the assay >10-fold for the AZD0530 majority of KRAS mutations. Results We likened the COLD-PCR improved melting curve solution to melting curve evaluation without COLD-PCR also to traditional Sanger sequencing. Within a cohort of 61 formalin-fixed paraffin-embedded colorectal tumor specimens 29 had been categorized as mutant and 28/61 as outrageous type across all strategies. Significantly 4 (6%) had been re-classified from outrageous type to mutant AZD0530 with the even more delicate COLD-PCR melting curve technique. These 4 examples were verified to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five indie mixing research using mutation-discordant pairs of cell lines and individual specimens demonstrated the fact that COLD-PCR improved melting curve assay could regularly detect right down to 1% mutant DNA within a outrageous type history. Conclusions We’ve created and validated a cheap rapid and extremely sensitive scientific assay for KRAS mutations this is the initial record AZD0530 of COLD-PCR coupled with probe-based melting curve evaluation. This assay improved diagnostic accuracy in comparison to traditional PCR and direct sequencing significantly. History KRAS (Kirsten rat sarcoma pathogen homolog 2) is certainly a membrane-anchored G-protein that works downstream from the epidermal development aspect receptor (EGFR) to activate pro-growth and anti-apoptotic pathways like the MAP kinase and PI3 kinase pathways [1]. Mutations in codons 12 and 13 from the KRAS gene confer level of resistance to drugs directed at EGFR by impairing GTPase activity which leads to constitutive EGFR-independent signaling. KRAS is certainly one of the most often mutated oncogenes in individual cancers with significant mutation prices in keeping epithelial malignancies such as for example cancer of the colon (~40%) lung tumor (~20%) gastric tumor (~10%) and pancreatic tumor (~65%) (COSMIC data source; http://www.sanger.ac.uk/genetics/CGP/cosmic/) [2 3 The function of KRAS mutation position in clinical decision building is most beneficial defined for colon cancer. Several large randomized-controlled trials exhibited no benefit from expensive anti-EGFR drugs such as cetuximab (Erbitux?) in patients with KRAS-mutant colon cancer [4 5 The National Comprehensive Malignancy Network (NCCN) now recommends KRAS testing prior to initiation of anti-EGFR therapy AZD0530 in colon AZD0530 cancer patients [6]. Accumulating evidence also suggests a role for KRAS testing to guide therapy in patients with non-small cell lung cancer [7 8 Greater than 95% of KRAS mutations occur in codon 12 or codon 13. Within these codons G12 D (GGT to GAT) G12V (GGT to GTT) and G13 D (GGC to GAC) comprise ~80% of the mutations [2 4 Less frequent mutations include G12 S (GGT to AGT) G12C (GGT to TGT) G12R (GGT to CGT) and G12A (GGT to GCT). Silent mutations are exceedingly rare http://www.sanger.ac.uk/genetics/CGP/cosmic/. CO-amplification at Lower Denaturation heat (COLD)-PCR is usually a recently described method to selectively amplify mutant alleles in a wild type background that does not require any additional instrumentation or reagents to implement [9]. Two forms of COLD-PCR are described: fast COLD-PCR and full COLD-PCR (reviewed in [10]). Fast COLD-PCR enriches G:C to A:T mutations that slightly but predictably lower the melting heat (Tm) of the PCR amplicon by using a crucial denaturation heat (Tc) that favors PCR amplification of the mutant allele. Full COLD-PCR theoretically enhances detection of any type of mutation via conditions which promote annealing of WT:mutant pairs and selective denaturation of these heteroduplexes at an empirically decided Tc. As the name implies fast COLD-PCR has the advantage of being more rapid than full COLD-PCR (1-2 hours of instrument time compared to 5-8 hours) and is also easier to troubleshoot and implement in our knowledge. Fast COLD-PCR is certainly ideal.