A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms
Figure 1
Detection of JAK2 V617F, MPL W515L, and MPL W515K mutations with a multiplex snapback primer system. Negative first-derivative (dF/dT) plot of melting curve consists of two melting regions. The stem-loop hairpin melting region for mutation discrimination (55–75°C) and the double-strand amplicon for DNA template amplification control (75–95°C). (a) Samples with the JAK2 V617F mutation (purple) showed a melting peak at 59.3°C, while the melting peaks at 63.2°C and 67.6°C indicated the presence of MPL W515K (ginger) and MPL W515L (blue) mutations, respectively. The amplification controls of JAK2 and MPL were presented by the melting peak at 81.1°C and 91.2°C, respectively. The mixture of three mutant allele-positive standards (red) generated all the three mutation melting peaks. In wild-type control sample (pink), only the double-strand control was amplified. (b) The amplicon melting curves of DNA from patients with JAK2 V617F, MPL W515L, MPL W515K, and concurrent JAK2 V617F and MPL W515K mutation.
