Mutations in Patients with Myelodysplastic Syndromes Machado-Neto JA et al. CLINICS 2011;66(5):793-799 Figure 2 - Fragment analysis of FLT3-ITD and NPM1 mutations. Representative fragment size analysis of a MDS patient with wild-type alleles for FLT3 (A), an AML patient with the FLT3-ITD mutation (B), an MDS patient with wild-type NPM1 (C) and an AML patient with a mutation in exon 12 of NPM1 (D). The arrows indicate the presence of the mutant allele. 5 minutes of denaturing at 94˚C followed by 35 cycles of 30 seconds at 92˚C, 30 seconds at 57˚C and 50 seconds at 72˚C, with a final step at 72˚C for 7 minutes. For RFLP analysis, JAK2 and FLT3 PCR products were digested with BsaXI or Eco321 (New England Biolabs, Hitchin, UK), respectively, according to the manufacturer’s protocol, and visualized on a 2.5% agarose gel. The normal genotype for JAK2 was represented by a 460-bp fragment, and the heterozygous genotype was represented by 460-bp, 241-bp and 189-bp fragments, whereas the homozygous mutant genotype produced 241-bp and 189-bp fragments. For FLT3-D835, the normal genotype was represented by 68-bp and 46-bp fragments, and the heterozygous genotype was represented by 114-bp, 68-bp and 46-bp fragments, whereas the homo- zygous mutant genotype produced only a 114-bp fragment. AML or PV patients with FLT3-D835 and JAK2 V617F mutations, respectively, were used as positive controls. Detection of the PI3K E542, E545 and H1047 mutations Screening for PI3K mutations was performed by sequen- cing PCR products. PCR was performed in a 50-mL reaction volume consisting of 100 ng of genomic DNA, 5 mL of 10X reaction buffer, 2 mL of 50 mM MgCl2, 2.5 units of Taq polymerase and 200 nM each of the forward and reverse primers (Table 3). The reaction conditions were set as follows: 5 minutes of denaturing at 94˚C followed by 35 cycles of 30 seconds at 94˚C, 50 seconds at 63˚C and 55 seconds at 72˚C, with a final step at 72˚C for 7 minutes. Sequencing reactions were performed in both directions with the ABI PRISM 796