Analysis of the reannealing- instead of melting-curve in the detection of JAK2 V617F mutation by HRM method
Authors Moradabadi A, Fatemi A, Noroozi-Aghideh A
Received 5 February 2019
Accepted for publication 10 July 2019
Published 22 July 2019 Volume 2019:10 Pages 235—241
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Amy Norman
Peer reviewer comments 3
Editor who approved publication: Dr Martin Bluth
Alireza Moradabadi,1 Ahmad Fatemi,2 Ali Noroozi-Aghideh1
1Department of Hematology, Faculty of Paramedicine, Aja University of Medical Sciences, Tehran, Iran; 2Department of Hematology, School of Allied Medical Sciences, Kerman University of Medical Sciences, Kerman, Iran
Background: The Janus kinase 2 (JAK2) has an important role in the intracellular signaling in normal and neoplastic cells. JAK2 mutation, called JAK2 V617F, is frequently found in Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess the analytical efficiency of high-resolution melting (HRM) method using reannealing-curve analysis in comparison with routine melting-curve analysis for JAK2 V617F mutation detection.
Method: Twenty-three samples including one negative synthetic standard DNA, two 50% and 75% positive synthetic standard DNA samples, five wild-type samples and 15 samples positive for JAK2 V617F were examined by HRM. Melting and reannealing stages were performed, and then, raw and normalized curves were compared between the two stages.
Results: In melting-curve analysis, the wild-type and mutant samples had different melting temperatures (75/53°C and 75/10°C, respectively). In normalized curves corresponding to reannealing method, mutant samples were better separated from the baseline than in melting method as well as for samples with different mutant DNA burden from each other. Furthermore, wild-type samples were more homogenous in the normalized curves corresponding to reannealing than in melting method. This means that patients with a low allelic burden may be wrongly interpreted as normal in the common melting method.
Conclusion: We suggest the use of reannealing instead of the melting-curve analysis for the detection of sequence variations, especially for large-scale mutation and allele burden measurement in clinical settings. However, more evaluations with more sample size will better improve the benefits of reannealing-curve analysis in research and clinic.
Keywords: HRM, melting, reannealing
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