Sequencing of TP53 in cisplatin-treated and cisplatin-resistant cells

First, A549 cells were cultured from 500 ng/mL of cisplatin and the concentration was increased to 800 ng/mL by continuous passaging to the point of drug resistance, repeating the previous operation (A549-c). In addition, there were untreated A549 and A549/DDP cells.Each sample was 1×106 cells before DNA extraction to ensure enough tissue to be processed. The primer pairs were: TP53-1,5'-GTCCCTCTCTGATTGTCTTTTCC-3' and 5'-ACTGACAGGAAGCCAAAGGG-3'; TP53-2,5'-TGTTTGTTTCTTTGCTGCCG-3' and 5'-CAAATAAGCAGCAGGAGAAAGC-3'; TP53-3,5'-GAGACCATCCTGGCTAACGG-3' and 5'-ACACCATCGTAAGTCAAGTAGCATC-3'. DNA was isolated by a Ezup Column Animal Genomic DNA Purification Kit. Samples were re-suspended in buffer CL, proteinase K, CW1 Solution, CW2 Solution, and CE Buffer for further purification and PCR. After DNA purity and concentration were determined, 1 µL template DNA, 2 µL primer sequences, and 2.5 µL of Taq Buffer (with MgCl2) (10×) were mixed together for the PCR cycle. The PCR conditions were set to 95 °C for 5 min and followed by 40 cycles of 95 °C for 30 sec, 58 °C for 30 sec, and 72 °C for 30 sec with a final extension at 72 °C for 10 min. PCR products of 5 µL of each were analyzed in 1% agarose gel with 500 and 1000 bp ladder DNA markers. The resulting PCR products were purified and sequenced in both directions using Sanger sequencing (Sangon Biotech Co., Ltd., Shanghai, China).