Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay

Hiraku Takei, Soji Morishita, Marito Araki, Yoko Edahiro, Yoshitaka Sunami, Yumi Hironaka, Naohiro Noda, Yuji Sekiguchi, Satoshi Tsuneda, Akimichi Ohsaka, Norio Komatsu

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    A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

    Original languageEnglish
    Pages (from-to)e104958
    JournalPLoS One
    Issue number8
    Publication statusPublished - 2014


    ASJC Scopus subject areas

    • Medicine(all)

    Cite this

    Takei, H., Morishita, S., Araki, M., Edahiro, Y., Sunami, Y., Hironaka, Y., Noda, N., Sekiguchi, Y., Tsuneda, S., Ohsaka, A., & Komatsu, N. (2014). Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay. PLoS One, 9(8), e104958.