RNA extraction and cDNA conversion
Total RNA was extracted from the peripheral blood samples and was reverse transcribed to cDNA using commercial available cDNA synthesis kit (Qiagen mini blood RNA extraction kit; Cat. No. 52304 and Verso cDNA synthesis kit; Cat. No. AB1453/A). The RNA extraction and cDNA preparation was performed according to the manufacturer’s instructions.
PCR amplification of BCR/ABL gene, Direct (Sanger) and Next generation sequencing
Semi nested pcr was performed to amplify kinase domain of BCR/ABL fusion gene. Direct sequencing was performed on amplified product using Big Dye Terminator chemistry (Applied biosystem, USA) in ABI 3130 automated DNA analyser . For Next generation sequencing sample was out sourced and it was performed by Medgenome India Pvt. Ltd.
The potential template for human ABL1 protein sequence was identified using BLAST-PDB search . Models were generated using commands like align2d.py and model-single.py wherein the former aligns the ABL1 protein sequence with the template structure and the latter helps in modelling the 3D structures based on the query-template combo alignment. Of the ten generated models, the structure with least Discrete Optimized Protein Energy (DOPE) was selected and considered for energy minimization using GROMACS . The ligand Imatinib was obtained from Protein Data Bank with a PDB id of 6HD4 and were separated from the protein using Swisspdbviewer[12,13]. Modelled structure was validated using ProSA-Web and Procheck-Ramachandran plot and later considered for docking studies . Two mutations at position 250 and 287 (residues got renumbered after modelling) were introduced within the wild type protein to generate three mutants. After mutations, they were termed as mut1 (Gly250Glu), mut2 (Ala287Tyr) and mut3 (gly250glu and Ala287Tyr). All three mutants were energy minimized using GROMACS.
Kollman charges were added to the modelled wild and mutant ABL1 protein and saved as .pdbqt file which were considered for docking with drugs like Imatinib, Dasatinib and Nilotinib. The prepared proteins and the ligand structures were considered for docking for which the grid box need to be generated. For the same, the active sites were much required which was obtained from the 3D structures available from Protein Data Bank. As per the PDB ids: 6HD4 and 6HD6 amino acids like Asp355, Glu260, Ile334, Thr289, Met 292, His335 (residues renumbered based on the modelled structures) interacts with imatinib drug molecule. Based on the drug binding site, the grid size was set to 82x 96x56 xyz points with grid spacing of 0.364 Å and grid center was designated at dimensions (x, y, and z): 5.367, 9.173 and 12.966.