CLINICS 2011;66(5):793-799 Table 3 - Primer sequences and restriction enzymes Mutations in Patients with Myelodysplastic Syndromes Machado-Neto JA et al. Gene FLT3 NPM1 JAK2 FLT3 PI3K PI3K Mutation ITD exon 12 V617F D835 exon 9 exon 20 Primers sequences F: 59-GCAATTTAGGTATGAAAGCCAGC-39 R: 59-CTTTCAGCATTTTGACGGCAACC-39(HEX) F: 59-GTGGTAGAATGAAAAATAGAT-39(FAM) R: 59-CTTGGCAATAGAACCTGGAC-39 F: 59-GGGTTTCCTCAGAACGTTGA-39 R: 59-TCATTGCTTTCCTTTTTCACAA-39 F: 59-CCGCCAGGAACGTGCTTG-39 R:59-GCAGCCTCACATTGCCCC-39 F: 59-TTACAGAGTAACAGACTAGC-39 R: 59-TTTTAGCACTTACCT GTGAC-39 F: 59-AGCTATTCGACAGCAGTGCC-39 R: 59-TTGTGTGGAAGATCCAATCC-39 Restriction enzyme site BsaXI Eco321 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 20 seconds at 92˚C, 30 seconds at 57˚C and 45 seconds at 72˚C, with a final step at 72˚C for 7 minutes. After dilution (1:20) in water, 1 mL of each PCR product was mixed with 9 mL of Hi- Di formamide (Applied Biosystems, Foster City, CA) and 0.5 mL of GeneScan 500-ROX size marker, and the mixture was denatured for 5 minutes at 95˚C. Samples harboring the mutation were identified based on the areas under the curves representing the wild-type (FLT3:397 bp and NPM1:294 bp) and mutated alleles (FLT3-ITD.397 bp and NPM1.294 bp). AML patients with FLT3-ITD or NPM1 mutations were used as positive controls. Detection of the JAK2 V617F and FLT3-D835 mutations Identification of JAK2 and FLT3 genotypes was performed using PCR-restriction fragment length polymorphism (PCR- RFLP) analysis. 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: Figure 1 - PCR and Sequencing of exons 9 and 20 of PI3K. The fragment size of the exon 9 (A) and exon 20 (B) of PI3K are indicated in the figure. In both figures A and B, lane 1: Ladder 100bp fragments; lane 2: negative control; lanes 3 and 4: amplicons obtained from genomic DNA of patient MDS patients (RA). Representative PI3K sequencing from MDS patients, determined by automated sequence analysis of exon 9 (C) and 20 (D). The localization of the most frequent hotspot mutations are highlighted in the figure. 795