FLT3 transcripts are expressed by human leukemia/lymphoma-derived cell lines and primary AML blasts. Total cellular RNA was extracted from leukemia/lymphoma cell lines and patients' blast cells. RNA (50 ng-100 ng) was reverse transcribed and amplified using primer pairs for FLT3 and actin. PCR products were resolved on 2.5% agarose gel in the presence of ethidium bromide, and photographs were taken under UV transillumination. If an FLT3/ITD mutation is present, a higher molecular weight band than wtFLT3 will be visualized. To detect mutations at D835, EcoRV digestion of the PCR-amplified region including the D835 and I836 codons was used prior to electrophoresis. These codons are encoded by the sequence GATATC, which forms the EcoRV restriction site. If this site is mutated, the PCR product will be resistant to digestion. (U, undigested; D, digested.) (A) Human leukemia/lymphoma derived cell lines. (B) Primary AML blasts. Patients 3, 6, and 7 expressed FLT3/ITD mutations. Patient 8 expressed a D835 mutation.