Open AccessDetection of the JAK2 mutation in myeloproliferative neoplasms by asymmetric PCR with unlabeled probe and high‐resolution melt analysis
Author(s) -
Cao HongCui,
Lin Jiang,
Qian Jun,
Yao DongMing,
Li Yun,
Yang Jing,
Chen Qin,
Chai HaiYan,
Xiao GaoFei
Publication year - 2011
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.20474
Subject(s) - high resolution melt , myeloid leukemia , mutation , jak2 v617f , mutant , microbiology and biotechnology , pathogenesis , medicine , polymerase chain reaction , cancer research , biology , gene , genetics , pathology
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. J. Clin. Lab. Anal. 25:300–304, 2011. © 2011 Wiley‐Liss, Inc.