Detection of the JAK2 V617F mutation by asymmetric PCR and melt curve analysis

Abstract

The chronic myeloproliferative disorders (CMPDs) are a heterogeneous group of clonal hematopoietic diseases characterized by production of increased numbers of mature leukocytes, erythrocytes, and/or platelets. Clinically these disorders are often insidious in onset, produce nonspecific thrombotic or hemorrhagic complications, and can be easily confused with a variety of benign, reactive conditions. Thus, confirming a CMPD can be difficult as it is often a diagnosis of exclusion. The recently identified JAK2^V617F mutation is frequently present in the classic CMPDs polycythemia vera, essential thrombocythemia, and chronic idiopathic myelofibrosis. JAK2^V617F determination has proven to be a useful diagnostic tool in patients with some clinical features suggestive for a CMPD, and may have benefit as a way to monitor known disease. There are several published molecular assays for the JAK2^V617F target, of variable sensitivity and technical complexity, many of which are not easily replicated in a typical clinical laboratory. We present a robust, sensitive PCR/melt curve assay for the JAK2^V617F mutation which uses the widely available Roche LightCycler® platform, and is thus applicable to many clinical molecular laboratories.