Fig. 1

Development of RFN-AS-PCR for detection of FLT3D835Y. a. Schematic illustration of the RFN-AS-PCR method. b and c The sensitivities of nested AS-PCR and RFN-AS-PCR methods were determined by using purified plasmid DNAs. Mixtures of FLT3 plasmid DNAs containing the indicated percentages of the FLT3D835Y mutant were amplified with primers F1_5 and F1_3. The PCR products were left undigested (b) or digested with restriction enzyme EcoRV (c) and then subjected to nested AS-PCR analyses with a primer mixture containing F1_5n, F1_3n, F1_mut, and F1_wt. The final PCR products were analyzed on 3% agarose gel, and DNA bands were visualized by staining with SYBR green. The positions of wild type FLT3- and mutant FLT3D835Y-specfic products are indicated. The higher molecular weight bands shared by both wild type FLT3 and mutant FLT3D835Y are products of primer pairs F1_5n/F1_3n (403 bp), F1_5/F1_3, F1_5n/F1_3, and/or F1_5/F1_3n