Detection of the JAK2(V617F) mutation in myeloproliferative disorders by melting curve analysis using the LightCycler system. Academic Article uri icon

Overview

abstract

  • CONTEXT: A specific mutation, JAK2(V617F), was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory. OBJECTIVE: We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing. DESIGN: Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind). RESULTS: The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2(V617F) amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2(V617F) was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia. CONCLUSIONS: These findings demonstrate the suitability of this assay for identifying JAK2(V617F) in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2(V617F) in archived specimens provides a new tool for studying the prognostic significance of this mutation.

publication date

  • July 1, 2006

Research

keywords

  • DNA Mutational Analysis
  • Molecular Diagnostic Techniques
  • Mutation
  • Myeloproliferative Disorders
  • Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins

Identity

Scopus Document Identifier

  • 33745966245

Digital Object Identifier (DOI)

  • 10.5858/2006-130-997-DOTJMI

PubMed ID

  • 16831057

Additional Document Info

volume

  • 130

issue

  • 7