Sequencing of TP53 in cisplatin-treated cells

A549 cells were cultured from low dose (10 μM), moderate dose(25 μM), and high dose(50 μM) cisplatin for 30 days by continuous passaging to the point of drug resistance, repeating the previous operation in H460 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).