title: Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation.
abstract: Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.
authors: Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors: Soji
authors: S
authors: Morishita
authors: Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan; Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors: Kochi
authors: K
authors: Takahashi
authors: Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors: Marito
authors: M
authors: Araki
authors: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors: Yumi
authors: Y
authors: Hironaka
authors: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors: Yoshitaka
authors: Y
authors: Sunami
authors: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors: Yoko
authors: Y
authors: Edahiro
authors: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors: Miyuki
authors: M
authors: Tsutsui
authors: Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors: Akimichi
authors: A
authors: Ohsaka
authors: Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
authors: Satoshi
authors: S
authors: Tsuneda
authors: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors: Norio
authors: N
authors: Komatsu
mesh_terms: D000483:Alleles; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009196:Myeloproliferative Disorders / Q000201:enzymology* / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D053614:Janus Kinase 2