Identification of JAK2V617F in normal blood samples. Blood cell DNAs from normal donors were analyzed by using the RFN-AS-PCR method. A. A typical analysis of multiple blood samples. Sample no. 5 was identified JAK2V617F positive. B. Verification of the positive samples by DNA sequencing. For this purpose, the product of first round PCR with primers P1 and P1r was either left untreated or digested with BsaXI before nested PCR with primers P2 and P2r. The nested PCR products were gel-purified and subjected to DNA sequencing analysis with primer P2r. Note that without BsaXI digestion DNA sequencing failed to reveal any mutant allele, but after restriction enzyme digestion, a clear mutant allele (base A in the indicated position) was detected.