Allele-specific PCR assays and DNA sequencing of the JAK2^V617F mutation. (A) PCR design and primer sequences. (B) Verification of allele-specific PCR analysis by using standard DNA plasmids. A 521-bp DNA fragment from genomic DNAs containing wild-type and JAK2^V617F mutation with P1 and P1r primers and cloned into the pBluescript KS vector (Stratagene, La Jolla, CA). A total of 4 ng plasmid DNA mixtures containing the indicated amount of JAK2^V617F mutant were subjected to PCR analysis with a primer mixture of P2, P2r, Pmr, and Pnf. (C-D) Allele-specific PCR analysis of blood genomic DNA. Genomic DNA was amplified by PCR with primers P1 and P1r and further amplified with allele-specific primer mixture P2, P2r, Pmr, and Pnf. PCR products were analyzed on 3% agarose and visualized with ethedium bromide staining. The expected PCR products were indicated by i (453 bp), ii (279 bp), and iii (229 bp). Among the blood DNA samples (C-D), nos. 787, 978, and 1385 are considered negative, and all the rest are considered positive. (E) Sequencing of PCR products from genomic DNAs amplified by using primers P1 and P1r. Sequencing was performed from the 3′-side with primer P1r. Percentages of the mutant in the total PCR products were determined by measuring the relative peak heights of DNA sequencing data in reference to those obtained with standard DNA mixtures containing known proportions of JAK2^V617F and wild-type JAK2.