A novel melting curve-based method for detecting mutations in acute myeloid leukemia.

The gene encodes a class III tyrosine kinase receptor. Specific somatic mutations in have been associated with acute myeloid leukemia (AML) and are markers of a poor prognosis in AML. Various methods have been used to detect the gene mutation; however, the suitability of these methods in the clinical management of AML remains unclear. The current study developed a novel method, using modified hybridization probes and melting curve analysis, for detecting mutations in exon 17. Dual-labeled self-quenched oligonucleotide probes containing two segments, labeled with carboxyrhodamine or hexachlorofluorescein, were designed to detect sequences around the D816 or N820/N822 mutation hot spots in exon 17 of . The exon 17 region of was amplified by polymerase chain reaction using control plasmids carrying wild-type or mutant sequences, or genomic DNA derived from AML patients. Melting curve analysis of the amplification products was performed using a self-quenched probe. The results showed that the detection sensitivity, assayed using mutation-positive control plasmids, was 10% for the N820G mutation and 5% for the six other mutations; N822K(A), N822K(G), D816V, D816Y, D816H and D816F. In addition, mutations were identified in six of the 12 samples from the core-binding factor (CBF)-AML patients. This demonstrates that the novel method developed in the present study, is simple, rapid, specific and highly sensitive, and may facilitate the diagnosis and treatment of CBF-AML.