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Updated: Jul 9, 2026
Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation
Published on: August 23, 2024
Detection of the JAK2V617F mutation by asymmetric PCR and melt curve analysis
Bobbie Collett Sutton1, Richard A Allen, Zhizhuang Joe Zhao
1Department of Pathology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
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.
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.
