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Background  Ph- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. JAK2V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2V617F can cause MPNs, and various methods have been developed to detect JAK2V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment.    Methods  In the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele, and 3) detection of JAK2V617F by allele-specific PCR with nested primers.    Results  We tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2V617F. We were able to detect JAK2V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found three JAK2V617F-positive cases, which would have remained undetected using a less sensitive method.    Conclusions  We have developed a highly sensitive method that will allow for detection of JAK2V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.

Development of a highly sensitive method for detection of JAK2V617F