Identification of the Jak2V617F mutation by direct DNA sequencing and dHPLC analysis in polycythemia vera. RNA was isolated and cDNA was prepared from the chronic myelogenous leukemia (CML) cell line K562, the erythroleukemia cell line HEL, and peripheral blood from patients with polycythemia vera, obtained with informed consent and according to institutional protocol approved by the Department of Clinical Medicine at Mannheim University of Heidelberg, per the Declaration of Helsinki. (A) Chromatograms of peripheral-blood samples from patients with polycythemia vera after direct sequencing of the forward strand of a PCR-amplified V617 proximal region in Jak2 is shown. The arrows indicate the position of the base implicated in the V617F substitution. The bottom panels show the expected mRNA and protein sequence in Jak2 (wild-type) and Jak2 with the V617F substitution. (B-C) A V617 proximal region within Jak2 was amplified by PCR, digested by Surveyor enzyme, and subjected to dHPLC analysis. Test samples (solid line) were compared to a control sample (dashed line). The arrows indicate the expected retention of the fragments in the presence of the V617F mutation. Chromatograms of peripheral-blood samples from patients with polycythemia vera (B) or HEL-cell samples indicated as a relative percentage of cells in a mixture with K562-cell samples (C) are shown.