Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera

Abstract

Background: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described.

Methodology/principal findings: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% 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 JAK2 V617F 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.

Figure 1. Unlabeled probe MCA on Rotor-Gene® Q.

Deivative (dF/dT) plot of melting curve consists of two melting regions. The probe-target melting region lies in left side. Samples with the T allele (green) had a lower Tm of 57.3°C, while samples harboring the G allele (red) showed a Tm of 63.6°C. Therefore, the G/T heterozygous samples (blue) manifested both melting peaks of these alleles. Each genotype followed a unique path that distinguished itself from the others.

Figure 2. Detecting sensitivity of unlabeled probe MCA on Rotor-Gene® Q.

For standard heterozygous samples containing over 3% (blue) T allele (mutation), there was a shape melting peak at Tm of the probe-mutation intermediates, which could be easily distinguishable from that of the wild type melting transition.

Figure 3. ARMS assay for JAK2 V617F.

Of tracks for each sample, bands in 229-bp suggested the existence of the wild type allele, while a mutant allele was indicated by the presence of a band in 279-bp. The 463-bp product served as a control of amplification. M, 2000 bp DNA ladder; 1, dd H₂O water control; 2, HEL cell line DNA as JAK2 V617F homozygous control; 3, RPMI82264 cell line DNA as wild type homozygous control; 4, Patient sample with no JAK2 V617F mutation; 5, Patient sample harboring wild type/JAK2 V617F positive heterozygote.

Figure 4. Validation of the unlabeled probe MCA on three different real-time instruments.

LightCycler® 480 (Figure 4A), ABI® 7500 real-time PCR system (Figure 4B) and Mastercycler® ep realplex (Figure 4C). The wild type homozygote (red) presented a probe melting peak of 63.8°C (LightCycler®), 61.7°C (ABI® 7500) and 63.4°C (Mastercycler®), while the JAK2 V617F homozygote (green) produced a probe melting peak of 57.9°C (LightCycler®), 55.6°C (ABI® 7500) and 57.5°C (Mastercycler®). A wild type/mutation heterozygote showed both melting peaks of wild type and mutation.