AS-PCR was performed using three primer pairs consisting of 1) T315I mutant primers, forward primer (MT_F) (5'-GCCCCCGTTCTATATCATAAT-3') and reverse primer (MT_R) (5'-GGATGAAGTTTTTCTTCTCCAG-3'), which was adapted from the previously published primer set [
20,
26], 2) the WT primers, WT_F (5'-TGGTTCATCATCATTCAACGGTGG-3') and WT_R (5'-GTTCCCGTAGGTCATGAACTCAG-3'), and 3) internal control primers, forward (β-actin_F) (5'-gtggggcgccccaggcacca-3') and β-actin_R (5'-gtccttaatgtcacgcacgatttc-3') [
27]. First, the AS-PCR was optimized by varying annealing temperature (Ta) (55° to 62°C), MgCl
2 concentration (1.0-2.5 mmol/L), and primer ratios (MT: WT ratio of 8:2, 7:3, 6:4, and 5:5). Briefly, the optimized condition was performed in a 25-μL mixture of 1 μL cDNA, 2.5 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 3% DMSO, and 0.625 unit of
Taq DNA polymerase (Invitrogen, USA) together with 14 pmol of MT primers, 6 pmol of WT primers, and 1 pmol of β-actin primers. The PCR profile was as follows: initial denaturation at 95°C for 5 minutes (min), followed by 35 cycles of denaturation at 94°C for 45 seconds (sec), annealing at 57°C for 30 sec, extension at 72°C for 1 min, and final extension at 72°C for 5 min. PCR products of T315I mutant, T315WT, and β-actin were 158 bp, 374 bp, and 540 bp, respectively. The products were assessed on a 2% agarose gel and staining with ethidium bromide. Thirty RNA sample from non-leukemic patients were used as negative control samples to optimize AS RT-PCR conditions for T315I.