To optimize the AS-PCR condition.2.2 Detection of T315I mutation by AS-PCRAS-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′-GGATGAAGTTTTT CTTCTCCAG-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′-GTTCCCGTAGGTCATGAACTCAG3′), and 3) internal control primers, forward (b-actin_F) (5′-gtggggcgccccaggcacca-3′) and b-actin_R (5′-gtc cttaatgtcacgcacgatttc-3′) [27]. First, the AS-PCR was optimized by varying annealing temperature (Ta) (55?to 62 ), MgCl2 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-L mixture of 1 L 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 b-actin primers. The PCR profile was as follows: initial denaturation at 95 for 5 minutes (min), followed by 35 cycles of denaturation at 94 for 45 seconds (sec), annealing at 57 for 30 sec, extension at 72 for 1 min, and final extension at 72 for 5 min. PCR products of T315I mutant, T315WT, and b-actinThe primary PCR step was performed using a pair of primers designed to cover BCR-ABL gene. Two micrograms of cDNA template were amplified in a total volume of 20 L with the following constituents, 0.2 U of AZD0156 custom synthesis high-fidelity DNA polymerase (PhusionTM, FINNZYME), 5X Phusion buffer, 2 mmol/L of MgCl 2 , 0.2 mmol/L of each dNTPs, 10 pmol of each primers (forward primers: B2A _f 5′-acagcattccgctgaccatcaataag-3′ and reverse primer: BA_r 5′-atggtccagaggatcgctctct-‘3) [8]. The reaction mixture was placed in a thermal cycler (Veriti, Applied Biosystems, CA) under the PCR profile as follows: initial denaturation at 98 for 30 sec, 35 cycles of amplification (at 98 for 10 sec, 60 for 30 sec, and 72 for 1 min 30 sec), and a final extension at 72 for 10 min. The product band of 1,643 bp (B2A2) or 1,719 bp (B3A2) was visualized on ethidium bromidestained 1.5 agarose gel. A secondary PCR step for amplification of KD amino acid codon 206-428 using the internal two primer pairs, were designed to amplify two partially overlapping fragments consisting of fragment 1 forward primers: abl_1F (5′-tggttcatcatcattcaacggtgg-3′) and reverse primers: abl_1R (5’-tctgagtggccatgtacagcagc-‘3), and fragment 2 forward primers: abl_2F (5′-tcatgacctacgggaacctc-3′) and reverse primers: abl_1R (5’-atactccaaatgcccagacg-‘3). The PCR reaction was performed in a volume of 50 L containing 2 L first round PCR product, 0.2 U of high-fidelity DNA polymerase, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27321907 1.5 mmol/L of MgCl2 , 0.2 mmol/L of each dNTPs, and 15 pmol of each primers. The PCR profile was as follows: initial denaturation at 98 for 30 sec, 35 cycles of amplification (98 for 10 sec, 60 for 30 sec, 72 for 40 sec), and final extension at 72 for 5 min. The fragment 1 (447 bp) and fragment 2 (333 bp) PCR products were assessed and prepared for further analysis by DHPLC and sequencing. Prior to DHPLC analysis, mutant products were mixed with WT in a 1:1 ratio and denatured by heating at 95 for 5 min followed by gradual cooling at 1 /min to 25 within 70 min in order to allow heteroduplex and homoduplex formation [28]. DNA were analyzed using a WAVE?nucleic acid fragment analysis system (Transgenomic Inc, Omaha, NA, USA) b.
