EGFR Gene Mutation Detection Protocol

In a 20 μl assay, 1X Emerald GT PCR master mix (Takara/Clontech, USA) was added, along with m13-tagged forward and reverse target primers (5 μM). Approximately 50 ng of template DNA is added, in a typical assay, and made up with distilled water. Primers for exons 18, 19, 20, and 21 of the EGFR gene (NCBI Genbank Accession ID: NM_005228.3) were synthesized (Merck-Sigma, Bangalore, India). Design and characterization of the primer sequences for both sequencing and HRM were obtained from a previously published literature [18 (link)]. Thermal cycler settings included an initial denaturation of 95 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 10 min. The amplicons were assessed using 2% Agarose gel (SeaKem® LE Agarose, Lonza, USA). The PCR products were then subjected to post-PCR clean up to remove residual primers and other enzyme proteins using HighPure® PCR product purification kit (Roche Molecular Diagnostics, Switzerland).