Figure 4

Increasing the assay specificity of the JAK2 V617F mutation-specific PCR. 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. Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification. B. Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification. C. Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification. D. Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.