Figure 2

The sensitivity of QuanTAS-PCR for quantifying JAK2 mutant alleles. The JAK2 mutation-specific qPCR shown in panel A and the JAK2 exon 9 reference qPCR shown in panel B were run simultaneously (raw data (not baseline corrected) are shown). A range of samples containing different percentages of mutant allele in a background of wild-type allele were tested. For simplicity, only selected dilutions of the entire standard curve (prepared as described in the Methods) are shown. A: JAK2 mutation-specific PCR. The 100% mutant control DNA (MUT 100%), the mutant in wild-type mixes (MUT/WT) 10%, 1% and 0.1% and the no template control (NTC) were run in triplicate. The mutant in wild-type mix MUT/WT 0.01% and the 100% wild-type control (WT 100%) were run in 10 replicates. One of the 10 WT 100% replicates (in blue colour) amplified very late (LinRegPCR-calculated Cq = 58.2) compared to a single copy at 56.2. This represents a typical false positive and the sample was considered negative for the V617F mutation. The NTC did not amplify, as expected. B: JAK2 exon 9 reference qPCR used for DNA input and normalisation. Each sample was run in triplicate. The graph shows that all samples had an equivalent number of JAK2 templates. The NTC did not amplify, as expected.