Analysis of the reannealing- instead of melting-curve in the detection of JAK2 V617F mutation by HRM method

Abstract

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.