Table 1.
Sequences of primers and unlabeled probe for MCA and ARMS.
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.
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.