Background: Epidermal growth factor receptor (EGFR) deletion mutations are associated with the development of nonsmall-cell lung cancer (NSCLC) and can serve as useful biomarkers.
Aim: In the present study, a novel assay for the detection of EGFR hotspot mutations was designed to be highly sensitive and practically false-positive-free to harness the potential of detecting such mutations as biomarkers early in the diagnosis of NSCLC. The new assay draws from the polymerase chain reaction (PCR) for amplification, blue-white screening for initial allele discrimination, and Sanger sequencing for mutation confirmation.
Results: Mutant plasmids were mixed with wild-type DNA in ratios from 1:10 to 1:1000, followed by PCR amplification, blue-white screening, and sequencing. Mutants were successfully sequence confirmed for mixtures at ratios of 1:300 and 1:1000, highlighting the assay's high sensitivity and low risk of false-positives due to confirmation by Sanger sequencing.
Conclusion: With high sensitivity and low false positives, the present assay is appealing as an aid in the early diagnosis of NSCLC through liquid biopsy. The highly customizable nature of the assay provides the possibility of applications in the early diagnosis of other cancer-related genes through nonsense-transformable mutations.