Figure 3

HRM as a quality control step. The use of an intercalating dye rather than a fluorescent probe to monitor the reaction allows the addition of an HRM step to identify non-specific amplification. Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate. A. JAK2 V617F mutation-specific PCR high resolution melting. The NTC amplification product melted at a temperature clearly different (higher) than that of the targeted sequence, showing this was a case of non-specific amplification. It is important to note that the WT 100% amplification deriving from non-specific annealing of the mutant-specific forward primer to wild-type templates cannot be distinguished by HRM. B. JAK2 exon 9 PCR high resolution melting. All PCR products show the same melting profile, indicating no presence of non-specific amplification products. C. JAK2 V617F mutation-specific PCR amplification (early experiment, no Q-Solution was used). One of the NTC replicates amplified at a Cq value of about 54.6 and multiple WT 100% replicates amplified at Cq values that ranged from 46.5 to 58. D. JAK2 exon 9 PCR amplification, performed to check for DNA input and for normalisation.