4.3. Genomic DNA Extraction and Detection of EGFR Mutations Using PCR and DNA Sequencing

Genomic DNA was extracted from NCI-H1975, HCC-827, and A-549 cells as well as patient tissues using DNAiso Reagent (Takara, Dalian, China), according to the manufacturer’s instructions. The DNA concentrations were measured using the 260/280 ratio obtained with a spectrophotometer and adjusted to 150 ng/μL. The samples were stored at −20 °C until use.

Detection of EGFR mutations in these three NSCLC cell lines and tissue samples was performed by PCR amplification with a PCR amplification kit (TransGen, Beijing, China), followed by DNA sequencing. The primers for amplification of EGFR 19Del(2) and L858R regions were the same as described in our previous study (Table 2). The PCR mixtures contained 2 μL of genomic DNA, 1 μL of each primer (100 μM), 25 μL of 2× EasyTaq^®PCR Supermix, and 21 μL of ddH₂O; the thermal cycling conditions were set to an initial denaturation step at 94 °C for 5 min and then 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72° C for 20 s, and a final extension at 72 °C for 5 min. The PCR products were then sequenced by using an ABI PRISM 3730 Genetic Analyzer^® (ABI, Austin, TX, USA), and the data were analyzed by using ABI PRISM SeqScape Software Version 5.2.0^® (ABI, Austin, TX, USA). The experiments were repeated three times.

PCR primers used to detect EGFR mutations

FP: Forward Primer, RP: Reverse Primer, Mut: mutation type, Wild: wild type.