Ultrasensitive biosensing of low abundance BRAF V600E mutation in real samples by coupling dual padlock-gap-ligase chain reaction with hyperbranched rolling circle amplification

Abstract

BRAF V600E mutation is an important drive gene mutation and biomarker for tumor diagnosis, monitoring and target treatment. In order to meet the needs of identifying low abundance BRAF V600E mutation from real clinical specimens, a real time fluorescent biosensing strategy was developed by perfectly integrating dual padlock-gap-ligase chain reaction (DP-gLCR) with hyperbranched rolling circle amplification (HRCA). A pair of padlock probes was designed to induce DP-gLCR by using single-base gap coupling with matched deoxyribonucleotides substrates, which significantly increased the specificity of mutation discrimination. A large amount of circularized padlock probes were produced and triggered subsequent HRCA, generating numerous dendritic double-stranded DNA (dsDNA) products for highly sensitive real time fluorescent biosensing. The designed biosensing strategy could detect as low as 200 zM mutation target and distinguish as low as 0.01% genomic DNA with B-type Raf kinase (BRAF) V600E mutation in 40 ng wild-type genomic DNA, which was equal to almost one copy of mutant genomic DNA. Moreover, the proposed method was successfully applied in the detection of BRAF V600E mutation from real clinical samples, proving great potential for ultrasensitive biosensing of low abundance somatic mutation for early cancer diagnosis and treatment.