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

    Research output: Contribution to journalArticle

    2 Citations (Scopus)

    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 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
    Volume10
    Issue number3
    DOIs
    Publication statusPublished - 2015 Mar 20

    Fingerprint

    melting
    Freezing
    Assays
    Melting
    Alleles
    alleles
    mutation
    Mutation
    polycythemia
    Amplification
    assays
    neoplasms
    Nucleic Acid Probes
    methodology
    Neoplasms
    Oligonucleotides
    Screening
    Polymerase Chain Reaction
    Polycythemia Vera
    Polycythemia

    ASJC Scopus subject areas

    • Medicine(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Agricultural and Biological Sciences(all)

    Cite this

    Morishita, S., Takahashi, K., Araki, M., Hironaka, Y., Sunami, Y., Edahiro, Y., ... Komatsu, N. (2015). Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation. PLoS One, 10(3), [e0122003]. https://doi.org/10.1371/journal.pone.0122003

    Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation. / Morishita, Soji; Takahashi, Kochi; Araki, Marito; Hironaka, Yumi; Sunami, Yoshitaka; Edahiro, Yoko; Tsutsui, Miyuki; Ohsaka, Akimichi; Tsuneda, Satoshi; Komatsu, Norio.

    In: PLoS One, Vol. 10, No. 3, e0122003, 20.03.2015.

    Research output: Contribution to journalArticle

    Morishita, S, Takahashi, K, Araki, M, Hironaka, Y, Sunami, Y, Edahiro, Y, Tsutsui, M, Ohsaka, A, Tsuneda, S & Komatsu, N 2015, 'Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation' PLoS One, vol. 10, no. 3, e0122003. https://doi.org/10.1371/journal.pone.0122003
    Morishita, Soji ; Takahashi, Kochi ; Araki, Marito ; Hironaka, Yumi ; Sunami, Yoshitaka ; Edahiro, Yoko ; Tsutsui, Miyuki ; Ohsaka, Akimichi ; Tsuneda, Satoshi ; Komatsu, Norio. / Melting curve analysis after T allele enrichment (MelcaTle) as a highly sensitive and reliable method for detecting the JAK2V617F mutation. In: PLoS One. 2015 ; Vol. 10, No. 3.
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    AU - Hironaka, Yumi

    AU - Sunami, Yoshitaka

    AU - Edahiro, Yoko

    AU - Tsutsui, Miyuki

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    AU - Tsuneda, Satoshi

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