Detection of somatic mutations in AML

Peripheral venous blood or bone marrow samples were collected from newly diagnosed AML patients. Genomic DNA was extracted using E.Z.N.A.^® SQ Blood DNA Kit II (Omega Bio-Tek company, USA) according to the manufacturer’s instructions and stored at − 80 °C until use. FLT3-ITD mutations were detected as described elsewhere [34]. Briefly, DNA fragment between the 14th and the 15th exons of FLT3 gene was amplified by polymerase chain reaction (PCR), and the PCR products were then electrophoresed through 2% agarose gels. The PCR product of 328 bp was from the FLT3 wildtype allele. NPM1 and DNMT3A R882 mutations were detected using pyrosequencing, and R882 mutant allele ratio was also calculated. In detail, DNA segments containing the 12th exon of NPM1 or the 23th exon of DNMT3A were amplified through PCR in a final reaction volume of 50 µL, which contained 41 μL sterile double-distilled water, 5 μL PCR buffer, 2 μL DNA, 1.5 μL dNTP, 0.5 μL DNA polymerase, and 0.05 nM of each primer. Thermal cycling procedure for PCR was as follows: degeneration at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 57 °C for 35 s and 72 °C for 30 s; a final extension at 72 °C for 7 min. After verification by agarose electrophoresis, the amplified fragments were analyzed by pyrosequencing on the PyroMark Q24 Advanced platform (Qiagen, Germany) with the pyrosequencing primers. Sequences of the primers were shown in Additional file 1: Table S1.