PCR analysis of FLT3-ITDs and TKD mutations.

(A) Agarose gel electrophoresis of PCR with (lanes k, l, m, p) and without FLT3-ITDs. Lane q contains DNA of a normal donor, and lane r contains the water control. (B) The same set of samples analyzed for the FLT3 TKD mutation. Lanes f, h, and k showed an undigested band, arguing for a mutation. All 3 mutations were confirmed by sequencing. (C) Results of direct sequencing of PCR products obtained from patient UPN 03-066, corresponding to lane f in panel B, and UPN 23-031, corresponding to lane h in panel B. Note the residual wt FLT3 sequence visible in both samples. (D) Genescan analysis of FLT3-ITDs using denaturing PAA-gel electrophoresis and fluorescence detection. The ratio given for each sample denotes the relative proportion of the AUC of mutant and wt FLT3 alleles (ie, AUC FLT3-ITD/AUC FLT3-wt). Sample UPN 08-029 shows 3 different FLT3-ITDs of 355, 358, and 397 bp.