title: Detection of the JAK2 mutation in myeloproliferative neoplasms by asymmetric PCR with unlabeled probe and high-resolution melt analysis.
abstract: BACKGROUND
  Several methods have been established to detect the JAK2 V617F mutation, a frequent event involved in the pathogenesis of myeloproliferative neoplasms (MPNs). High-resolution melt (HRM) analysis is a newly established technique without the requirement of any gel-based post-PCR handling.
  
  
  METHODS
  An asymmetric PCR with unlabeled specific probe was developed and combined to HRM analysis o screen for JAK2 V617F mutation.
  
  
  RESULTS
  Heterozygous mutation was easily distinguished from homozygous JAK2 for the obvious shape change. Homozygous JAK2 mutant can be also well separated from wild-type JAK2 in the presence of internal temperature calibrators. The easily recognizable and maximal sensitivity of HRM analysis was 5% for the detection of JAK2 V617F mutation, higher than 25% of direct sequencing. In the test of blind screening of 223 samples (111 Ph- MPNs, 60 Ph+ chronic myeloid leukemia, and 52 acute myeloid leukemia), JAK2 V617F mutations were found in 78 (70%) patients with MPNs, but in none with chronic and acute myeloid leukemia. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing.
  
  
  CONCLUSIONS
  The HRM method with unlabeled probe could be used as convenient, sensitive and reliable diagnostic test for detection of JAK2 V617F mutation.
authors.affiliation: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People's Republic of China.
authors.forename: Hong-Cui
authors.initials: HC
authors.lastname: Cao
authors.forename: Jiang
authors.initials: J
authors.lastname: Lin
authors.forename: Jun
authors.initials: J
authors.lastname: Qian
authors.forename: Dong-Ming
authors.initials: DM
authors.lastname: Yao
authors.forename: Yun
authors.initials: Y
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.forename: Qin
authors.initials: Q
authors.lastname: Chen
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Chai
authors.forename: Gao-Fei
authors.initials: GF
authors.lastname: Xiao
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D007951:Leukemia, Myeloid / Q000201:enzymology / Q000235:genetics; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D004273:DNA, Neoplasm; C507924:JAK2 protein, human; D053614:Janus Kinase 2