title: A novel melting curve-based method for detectingc-kitmutations in acute myeloid leukemia.
abstract: Thec-kitgene encodes a class III tyrosine kinase receptor. Specific somatic mutations inc-kithave been associated with acute myeloid leukemia (AML) and are markers of a poor prognosis in AML. Various methods have been used to detect thec-kitgene 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 detectingc-kitmutations 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 ofc-kit. The exon 17 region ofc-kitwas 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,c-kitmutations 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.
authors: Department of Hematology, Zhongshan Hospital of Xiamen University, Xiamen, Fujian 361004, P.R. China.
authors: Quanyi
authors: Q
authors: Lu
authors: Department of Hematology, Zhongshan Hospital of Xiamen University, Xiamen, Fujian 361004, P.R. China.
authors: Xiao
authors: X
authors: Huang
authors: Department of Hematology, Zhongshan Hospital of Xiamen University, Xiamen, Fujian 361004, P.R. China.
authors: Huaying
authors: H
authors: Chen
authors: Department of Hematology, Zhongshan Hospital of Xiamen University, Xiamen, Fujian 361004, P.R. China.
authors: Xiaomin
authors: X
authors: Zhao
keywords: c-kit; genetic mutation; leukemia; melting curve; mutation detection