Figure 1

JAK2 real-time PCR melting curve. A representative JAK2 real-time PCR melting curve is shown. Serial dilution of a homozygous JAK2^V617F human erythroid leukemia cell line (HEL) with a homozygous JAK2 wild-type multiple myeloma cell line (RPMI8226) demonstrates the semiquantitative measurement of allele frequency by comparison of relative peak heights (47°C for JAK2^V617F compared with 55°C for wild-type). JAK2^V617F to wild-type ratio (HEL:RPMI8226) is indicated by light blue=1:0, light green=1:1, red=1:4, orange=1:9, brown=1:19, dark green=1:39 and dark blue=0:1. The negative control (water only) is shown in gray. As demonstrated in the brown curve with a barely observable JAK2^V617F peak, the lower limit of detection for this assay is one homozygous mutant cell in total 20 cells. The relative peak height of the JAK2^V617F allele decreases as the ratio of mutant to wild-type cells decreases, providing a semiquantitative measurement of allele frequency.