Molecular Detection of EGFR Mutation in Lung Cancer Using Bioinformatics Database with PCR Technique

Abstract

The aim of this study was to design a new model of polymerase chain reaction (PCR) based diagnostic method using mouse genome for one rare epidermal growth factor receptor (EGFR) gene mutations with diagnostic and prognostic values in lung cancer (this mutation are not covered by commercial test kits). From the systematic database search, one rare mutations identified which is within the tyrosine kinase (TK) domain of EGFR, with an insertion mutation in exon 20 namely A763_Y764insFQEA. For designing primers to detect the targeted regions surrounding the rare mutations, Primer3 Plus was carried out to design two sets of PCR primers for exon 20 of mouse EGFR gene. UCSC (University of California Santa Cruz) in silico PCR testing along with BLASTN search were used for primer specificity in terms of predicted target location (chromosome 11 for exon 20) and predicted amplicon sizes (276 bp for exon 20). PCR was partially optimised for the exon 20 with the presence of expected amplicon bands, along with unspecific and primer-dimer bands. The amplicon was sequenced and revealed the presence of the mutation. The mutation of interest selected was A763_Y764insFQEA (insertion of phenylalanine, glutamine, glutamic acid and alanine in between codon 763 and 764) in exon 20, the mutation was in the kinase domain of EGFR. The Identification and Analysis for rare