Pyrosequencing assay for detection of 1849G>T JAK2 mutation. (A) Examples of pyrosequencing results. The sequence read is (G/T)TCTGTGG. The mutation site with the adjacent T nucleotide is shaded. Peak heights are proportional to the amount of nucleotide present in the sequenced DNA. (top panel) Results from healthy control showing equal heights of the 1849G and the following T peak. The last peak is of double height, reflecting 2 adjacent G nucleotides. (middle panel) Ph^–CML, with the normal G allele constituting 40% and the mutant T allele 60% of the total amplified DNA. (bottom panel) HEL cell line showing 0% of normal G allele and 100% of mutant T allele. The height of the T peak is 200% (100% from the mutant allele and 100% from the adjacent T allele). (B) Reproducibility of quantitative analysis of the JAK2 mutation. Two independent PCR and pyrosequencing reactions were performed to quantify the presence of A on the antisense strand (x-axis) and T on the sense strand (y-axis). Correlation between the assays was nearly perfect (r² = 0.99). (C) Titration experiments showed linearity of the pyrosequencing assay. Healthy control DNA was mixed with 10%, 25%, 50%, and 75% DNA from a patient with MF carrying 50% of the mutant allele (○, broken line) or from a patient with PV whose DNA contained 80% of the mutant allele (•, solid line). Linear regression showed slopes of 0.47 and 0.77 and correlation coefficients (r²) of 0.98 and 0.99, respectively. (D) JAK2 gene is amplified in HEL erythroleukemic cell line. Healthy control DNA mixed with 25%, 50%, and 75% HEL DNA showed 40%, 67%, and 85% of mutant allele (○), indicating that the HEL cell line carries 4 mutant alleles and no normal allele. After correction for copy number, the dilution experiment showed a straight line (•, solid line).