Fluorometric Detection of Low-Abundance EGFR Exon 19 Deletion Mutation Using Tandem Gene Amplification

Abstract

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of lowabundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCRamplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review

“…Similarly, Kim et al also described a method for detecting low-abundance EGFR exon 19-del mutant genomic DNA: generation of long segments containing G-quadruplex structures in RCA by thioflavin T detection based on the fluorescence intensity of single-stranded DNA. This method detected as low as 0.01% of mutant genes in pooled normal plasma [ 42 ]. Chung et al used RCA combined with surface-enhanced Raman scattering to detect multiple point mutations in the KRAS gene.…”