Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation

Soji Morishita, Kochi Takahashi, Marito Araki, Yumi Hironaka, Yoshitaka Sunami, Yoko Edahiro, Miyuki Tsutsui, Akimichi Ohsaka, Satoshi Tsuneda, Norio Komatsu

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6 Citations (Scopus)


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 JAK2 V617F 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 Melca-Tle 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.

Original languageEnglish
Article numbere0122003
JournalPloS one
Issue number3
Publication statusPublished - 2015 Mar 20

ASJC Scopus subject areas

  • General Biochemistry,Genetics and Molecular Biology
  • General Agricultural and Biological Sciences
  • General


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