We developed ARMS-PCR to identify IDH2

R140Q mutation and endonuclease restriction analysis to identify DNMT3A R882H mutations; both these methods are rapid and easy to use and interpret. Thus, these methods can be used to verify unclear results obtained using HRM analysis. In addition, these methods provide a possibility to identify the most common mutations in DNMT3A and IDH2 in laboratories that do not have HRM-competent real-time PCR cyclers at their disposal. Secondary endonuclease restriction has higher sensitivity than HRM analysis that allows earlier identification of mutations at relapse during follow-up analysis [33]. For future applications this assay could also be adapted to the quantitative PCR (qPCR) technique. The forward primer can be modified to amplify only selleckchem the genomic Selleck CYC202 region containing the restriction Selleck LB-100 position that is lost in the mutated state, thus

allowing the exclusion of wt and mutated alleles as well as the quantitative assessment of DNMT3A mutation. The main characteristics of all the methods analysed in this study are summarised in Table 1. The measured sensitivities depend on assay conditions and equipment. For example, small amounts of non-specific amplicons and different salt or inhibitory concentrations can influence assay sensitivity [34, 35]. Therefore, each laboratory should validate the presented methods with their equipment before application. Both HRM analysis and ARMS-PCR had only low sensitivity, which possibly could lead to false-negative results. Therefore, low mutational ratios could be overlooked and these patients would receive an imprecise laboratory Pomalidomide diagnostic report. Potential reduction of amplicon size for both HRM and ARMS analyses could optimise sensitivities [36]. Moreover, adaption of the qualitative endonuclease restriction assay to a quantitative assay could further increase sensitivity and provide objective measurements of mutated cells [37]. In the future, sensitivity limitations for

screening DNMT3A and IDH1/2 mutations can be overcome by using allele-specific next-generation sequencing (NGS). This method provides high multiplexing possibilities together with high sensitivity and broad spectrum of detected mutations [38]. However NGS is associated with high costs, high hands-on time and high computational expertise. Because standardisation and validation of NGS can be challenging establishment of this method is an ongoing process in laboratory routine [39]. Conventional PCR-based methods are easy to standardise and validate and therefore could be used when NGS is being implemented in order to provide routine mutational screening of patients with AML.