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Identification of the JAK2 V617F Mutation in Chronic Myeloproliferative Disorders Using FRET Probes and Melting Curve Analysis

Gurunathan Murugesan PhD, Samer Aboudola MD, Hadrian Szpurka MS, Mary Ann Verbic MT(ASCP)SI, Jaroslaw P. Maciejewski MD, Raymond R. Tubbs DO, Eric D. Hsi MD
DOI: http://dx.doi.org/10.1309/TK0XL917XK2VLRPQ 625-633 First published online: 1 April 2006


We developed and validated a real-time polymerase chain reaction assay using fluorescent hybridization probes and melting curve analysis to identify the JAK2V617F mutation, which is implicated in a substantial proportion of chronic myeloproliferative disorders (CMPDs). DNA from 161 samples was isolated from peripheral blood granulocytes and formalin-fixed bone marrow clot sections in patients with CMPDs and without myeloproliferative disorders previously genotyped for the JAK2V617F (G→T) mutation, which included 114 wild types (GG) and 47 mutants (GT and TT). Melting curve analysis of these samples yielded 114 wild types, 42 heterozygotes, and 5 homozygotes showing 100% concordance. Analytic sensitivity of the assay for mutant DNA was 5% for the LightTyper (Roche Applied Sciences, Indianapolis, IN) and 10% for the LightCycler (Roche Applied Sciences). Consistent with earlier reports, 78% of the non–chronic myelogenous leukemia CMPD patients and 8% of non-CMPD patients displayed this mutation. This study demonstrates that clinical genotyping of the JAK2V617F mutation can be performed by melting analysis using both freshly isolated and formalin-fixed tissues.

Key Words:
  • JAK2
  • Mutation
  • Chronic myeloproliferative disorders
  • CMPD
  • Fluorescence resonance energy transfer
  • FRET probes
  • Melting curve
  • Real-time PCR
  • LightCycler
  • LightTyper