Figure 1

Development of restriction fragment nested allele-specific PCR (RFN-AS-PCR), an improved method for detection of JAK2V617F. A. Schematic illustration of the RFN-AS-PCR method. B and C. The sensitivities of the nested AS-PCR and RFN-AS-PCR methods were determined by using purified plasmid DNAs. Mixtures of JAK2 plasmid DNAs containing the indicated percentages of the JAK2V617F mutant were amplified with primers P1 and P1r. The PCR products were left undigested (panel B) or digested with restriction enzyme BsaX1 (panel C) and then subjected to nested AS-PCR analyses with a primer mixture containing P2, P2r, Pmr, and Pnf. The final PCR products were analyzed on 3% agarose gel, and DNA bands were visualized by staining with ethidium bromide. The positions of wild type JAK2 and mutant JAK2V617F are indicated.