Semiquantitative estimation of the MPL 1544 G>T and MPL 1543 TG>AA/total MPL ratio using sequencing and real-time PCR assay. One hundred percent mutated DNA was mixed in various proportions with 100% normal control DNA. The sequence traces and real-time PCR amplification plots indicating mutated (W515L or W515K) and wild-type MPL amplification plots for each dilution are shown, demonstrating the correlation of the techniques. The sensitivity of the sequencing approach was 5% to 10%, whereas the sensitivity of the real-time PCR assay was approximately 2% to 3% of mutated allele. However, because the purity of the lymphoid cell populations studied in this work was 98%, results lower than 2% of mutated allele were arbitrarily considered as negative because 2% of mutated myeloid cells could contaminate the samples. Semiquantitative results were expressed as shown in the center part of the figure.