Application Of Melting Curve Analysis Derived Techniques:Detection Of JAK2V617F Mutation In Myeloproliferative Neoplasms

Part1Development and Inter-Laboratory Validation of Unlabeled Probe Melting Curve Analysis for Detection of JAK2V617F Mutation in Polycythemia VeraObjective To develop a melting curve analysis system with a3’-C3blocked unlabeled probe for the identification of JAK2V617F mutation in the peripheral blood of polycythemia vera patients and validate the system on the routine genetic testing instruments.Methods Asymmetric PCR for detection of JAK2V617F with a3’-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene Q real-time cycler to establish the methodology. Standards with0%,1%,3%,5%,10%,25%,50%and100%mutation load were prepared with serial dilution of20ng/ul RPMI8226and HEL cell line DNA to determine the analytical sensitivity of this system. Intra-and inter-assay CVs of the melting temperature of both the mutation and wild-type probe-DNA heteroduplex were utilized to assess the analytical precision of the system with the mutation board-positive standard. We compared this method to the existing amplification refractory mutation systems and direct sequencing with the peripheral blood DNA samples from40patients with polycythemia vera and40healthy control subjects. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems, the LightCycler480, PRISM7500and Mastercycler ep realplex in two different laboratories.Results The unlabeled probe melting analysis could genotype JAK2V617F mutation explicitly with a3%mutation load detecting sensitivity. With an inter-and intra assay CV of3.14%/3.55%and1.72%/1.29%respectively, the melting temperature of the mutation and wild type probe-DNA heteroduplex covered the melting temperature of57.3±0.7℃and63.6±1.1℃respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments.Conclusions With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2V617F mutation than conventional methodologies. Verified with the favorable inter-and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.Part2A Snapback primer system for enrichment and detection of JAK2V617F Mutation in Myeloproliferative NeoplasmsObjective To develop a snapback primer based rare allele enrichment PCR system for the melting analysis detection of JAK2V617F mutation in the peripheral blood of myeloproliferative neoplasms patients.Methods Asymmetric PCR for detection of JAK2V617F with excessive5’-snapback primer, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene Q real-time cycler to establish the method. Standards with0%,0.01%,0.1%,1%,10%and100%mutation load were prepared with serial dilution of20ng/ul genomic DNA of RPMI8226and HEL cell line to determine the analytical sensitivity. Intra-and inter-assay CVs of the snapback primer-target single strand DNA melting temperature were employed to determine the analytical precision of the system with the mutation board-positive standard. The mutation detecting repeatability was evaluated with duplication of both the board-positive and wild type control. The methodology was compared to the unlabeled probe melting system and direct sequencing with the peripheral blood DNA samples from80myeloproliferative neoplasms patients and40healthy controls.Results The snapback primer system could enrich JAK2V617F mutation robustly for the subsequent melting curve analysis with a0.1%mutation load detecting sensitivity. With an inter-and intra assay CV of1.47%and2.51%respectively, the melting temperature of the mutation primer-DNA heteroduplex possessed a melting temperature of67.3±0.9℃. With the mutation enrichment ability, the snapback primer system can identify the samples with false-negative results in the conventional method such as unlabeled probe melting and direct sequencing.Conclusions Snapback primer PCR is a vigorous system for both the enrichment and detection of the rare allele JAK2V617F mutation with a high detecting sensitivity. As the whole procedure of the reaction is carried out in the closed tube, this method will enjoy a promising future for the molecular diagnosis of myeloproliferative neoplasms.