Abstract
BackgroundJAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described.Methodology/Principal FindingsAsymmetric PCR for detection of JAK2 V617F with a 3′-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments.ConclusionsWith a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.
Highlights
Somatic mutations of the JAK2 gene at V617F, which leads to constitutive JAK2 phosphorylation and kinase activation, occurs in almost all cases of polycythemia vera and approximately 50% of cases of chronic idiopathic myelofibrosis [1,2,3,4]
Because identification of the V617F mutation will be of potential use in the diagnosis, prognosis, and perhaps selection of treatment for myeloproliferative neoplasmas, many tools have been developed to genotype JAK2 V617F, including techniques such as restriction fragment length polymorphism (RFLP) [2], amplification refractory mutation system (ARMS) [7], real-time PCR [8,9] and direct sequencing [10,11]
Homozygous mutant (JAK2 V617F/JAK2 V617F) human erythroleukemia (HEL) and homozygous wild type multiple myeloma (RPMI8226) cell lines were purchased from the cell bank of type culture collection of Chinese Academy of Sciences and used as positive and negative controls, respectively
Summary
Somatic mutations of the JAK2 gene at V617F, which leads to constitutive JAK2 phosphorylation and kinase activation, occurs in almost all cases of polycythemia vera and approximately 50% of cases of chronic idiopathic myelofibrosis [1,2,3,4]. Because identification of the V617F mutation will be of potential use in the diagnosis, prognosis, and perhaps selection of treatment for myeloproliferative neoplasmas, many tools have been developed to genotype JAK2 V617F, including techniques such as restriction fragment length polymorphism (RFLP) [2], amplification refractory mutation system (ARMS) [7], real-time PCR [8,9] and direct sequencing [10,11] Some of these methods have limitations in sensitivity or in cost. JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms Various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described
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